Polypeptides having nucleic acid binding activity and compositions and methods for nucleic acid amplification

ABSTRACT

Polypeptides having nucleic acid binding activity are provided. Methods of using polypeptides having nucleic acid binding activity are provided. Fusion proteins and methods of using fusion proteins are provided. Fusion proteins comprising a polymerase and a nucleic acid binding polypeptide are provided. Fusion proteins comprising a reverse transcriptase and a nucleic acid binding polypeptide are provided. Methods are provided for amplifying a nucleic acid sequence using a fusion protein comprising a nucleic acid binding polypeptide and a polymerase. Methods are provided for amplifying a nucleic acid sequence using a fusion protein comprising a nucleic acid binding polypeptide and a reverse transcriptase.

This application claims the benefit of U.S. Provisional Application No.60/641,987, filed Jan. 6, 2005; and U.S. Provisional Application No.60/699,975, filed Jul. 15, 2005.

I. FIELD

Polypeptides having nucleic acid binding activity are provided. Methodsof using polypeptides having nucleic acid binding activity are provided.Fusion proteins and methods of using fusion proteins are provided.Fusion proteins comprising a polymerase and a nucleic acid bindingpolypeptide are provided. Fusion proteins comprising a reversetranscriptase and a nucleic acid binding polypeptide are provided.Methods of using fusion proteins to increase the efficiency of primerextension reactions, such as PCR, are provided. Methods of perfoming PCRusing rapid amplification cycles are provided.

II. INTRODUCTION

Polypeptides with nucleic acid binding activity are present in lowerorganisms, such as archaea, and higher organisms, such as eukaryotes.See, e.g., Pereira et al. (1997) Proc. Nat'l Acad. Sci. USA94:12633-12637; and Motz et al. (2002) J. Biol. Chem. 277:16179-16188.Polypeptides with nucleic acid binding activity have various functions.For example, certain polypeptides with nucleic acid binding activity,such as histones and histone-like proteins, are involved in thepackaging of chromatin into higher order structures. See, e.g., Pereiraet al. (1997) Proc. Nat'l Acad. Sci. USA 94:12633-12637. Certain otherpolypeptides with nucleic acid binding activity may play a role asprocessivity factors in DNA replication. See, e.g., Motz et al. (2002)J. Biol. Chem. 277:16179-16188.

Various methods can be used to amplify nucleic acids. One commonly usedmethod is the polymerase chain reaction (PCR). See, e.g., U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159. PCR typically comprises multiplecycles in which nucleic acid synthesis is initiated from at least twoprimers annealed to opposite strands of a target nucleic acid. Thisprocess allows exponential amplification of the target nucleic acid.

III. SUMMARY

In certain embodiments, a method of amplifying a nucleic acid sequenceis provided. In certain embodiments, the method comprises subjecting areaction mixture to at least one amplification cycle, wherein thereaction mixture comprises a double-stranded nucleic acid, at least twoprimers capable of annealing to complementary strands of thedouble-stranded nucleic acid, and a fusion protein comprising athermostable DNA polymerase and a nucleic acid binding polypeptide. Incertain embodiments, the at least one amplification cycle comprisesdenaturing the double-stranded nucleic acid, annealing the at least twoprimers to complementary strands of the denatured double-strandednucleic acid, and extending the at least two primers.

In certain embodiments, the time to complete one amplification cycle is20 seconds or less. In certain embodiments, the time to complete oneamplification cycle is 15 seconds or less. In certain embodiments, thetime to complete one amplification cycle is 10 seconds or less.

In certain embodiments, the annealing occurs at an annealing temperaturethat is greater than the predicted Tm of at least one of the primers. Incertain embodiments, the annealing temperature is at least about 5° C.greater than the predicted Tm of at least one of the primers. In certainembodiments, the annealing temperature is at least about 10° C. greaterthan the predicted Tm of at least one of the primers. In certainembodiments, the annealing temperature is at least about 15° C. greaterthan the predicted Tm of at least one of the primers. In certainembodiments, the annealing temperature is from about 62° C. to about 75°C. In certain embodiments, the annealing temperature is from about 65°C. to about 72° C.

In certain embodiments, the extending occurs at the annealingtemperature. In certain embodiments, the reaction mixture is held at theannealing temperature for 1 second or less.

In certain embodiments, the denaturing occurs at a denaturingtemperature that is sufficient to denature the double-stranded nucleicacid. In certain embodiments, the denaturing temperature is from about85° C. to about 100° C. In certain embodiments, the reaction mixture isheld at the denaturing temperature for 1 second or less. In certainembodiments, the reaction mixture is held at the denaturing temperaturefor 1 second or less and the annealing temperature for 1 second or less.In certain embodiments, the denaturing comprises bringing the reactionmixture to the denaturing temperature without holding the reactionmixture at the denaturing temperature after the denaturing temperatureis reached, and bringing the reaction mixture to the annealingtemperature without holding the reaction mixture at the annealingtemperature after the annealing temperature is reached.

In certain embodiments, the nucleic acid binding polypeptide comprisesan amino acid sequence of a nucleic acid binding polypeptide from athermophilic microbe. In certain embodiments, the nucleic acid bindingpolypeptide comprises an amino acid sequence of a nucleic acid bindingpolypeptide from Sulfolobus. In certain embodiments, the nucleic acidbinding polypeptide is a Crenarchaeal nucleic acid binding polypeptide.In certain embodiments, the nucleic acid binding polypeptide comprises asequence selected from: a) SEQ ID NO:20, b) a sequence having at least80% identity to SEQ ID NO:20, c) SEQ ID NO:6, d) a sequence having atleast 80% identity to SEQ ID NO:6, e) SEQ ID NO:1, and f) a sequencehaving at least 80% identity to SEQ ID NO:1.

In certain embodiments, the thermostable DNA polymerase comprises anarchaeal family B polymerase or a fragment or variant of an archaealfamily B polymerase having polymerase activity. In certain embodiments,the thermostable DNA polymerase comprises Pfu polymerase or a fragmentor variant of Pfu polymerase having polymerase activity.

In certain embodiments, the reaction mixture further comprises apolypeptide having 5′ to 3′ exonuclease activity.

In certain embodiments, the thermostable DNA polymerase comprises abacterial family A polymerase or a fragment or variant of a bacterialfamily A polymerase having polymerase activity. In certain embodiments,the thermostable DNA polymerase comprises Taq DNA polymerase or afragment or variant of Taq DNA polymerase having polymerase activity. Incertain embodiments, the thermostable DNA polymerase comprises afragment of Taq DNA polymerase lacking 5′ to 3′ exonuclease activity. Incertain embodiments, the thermostable DNA polymerase comprises acold-sensitive mutant of Taq polymerase. In certain embodiments, thethermostable DNA polymerase comprises a variant of Taq DNA polymerasehaving increased processivity relative to naturally occurring Taq DNApolymerase.

In certain embodiments, the reaction mixture further comprises anindicator molecule that indicates the amount of nucleic acid in thereaction mixture.

In certain embodiments, the reaction mixture further comprises anindicator probe capable of selectively hybridizing to a strand of thedouble-stranded nucleic acid. In certain embodiments, the indicatorprobe is a 5′-nuclease probe comprising a signal moiety capable ofproducing a detectable signal, and wherein extension of at least one ofthe at least two primers results in cleavage of the 5′-nuclease probe.In certain embodiments, cleavage of the 5′-nuclease probe increases thedetectable signal from the signal moiety.

In certain embodiments, the indicator probe comprises ahybridization-dependent probe. In certain embodiments, thehybridization-dependent probe is a hairpin probe comprising a signalmoiety capable of producing a detectable signal. In certain embodiments,hybridization of the hairpin probe to a strand of the double-strandednucleic acid increases the detectable signal from the signal moiety.

In certain embodiments, the method further comprises detecting theabsence or presence of an extension product from at least one of the atleast two primers during at least one of the at least one amplificationcycle.

In certain embodiments, the reaction mixture is subjected to up to 25amplification cycles. In certain embodiments, the reaction mixture issubjected to up to 30 amplification cycles. In certain embodiments, thereaction mixture is subjected to up to 40 amplification cycles.

In certain embodiments, the number of amplified molecules produced in atleast one of the at least one amplification cycle is from 1.6-fold to2-fold the number of molecules present at the start of the at least oneof the at least one amplification cycle. In certain embodiments, theamplification efficiency of the fusion protein in at least one of the atleast one amplification cycle is from 0.8 to 1.0.

In certain embodiments, a method of stabilizing an DNA:RNA duplex isprovided, wherein the method comprises combining the DNA:RNA duplex witha polypeptide comprising an amino acid sequence of a nucleic acidbinding polypeptide or a fragment thereof having nucleic acid bindingactivity.

In certain embodiments, a method of promoting the annealing ofcomplementary DNA and RNA strands is provided, wherein the methodcomprises combining the complementary DNA and RNA strands with apolypeptide comprising an amino acid sequence of a nucleic acid bindingpolypeptide or a fragment thereof having nucleic acid binding activity.

In certain embodiments, a method of generating DNA from an RNA templateis provided, wherein the method comprises exposing the RNA template toat least one primer and a fusion protein comprising a nucleic acidbinding polypeptide and a polymerase, wherein the polymerase is a familyB polymerase, a fragment of a family B polymerase, or a polypeptidehaving at least 80% identity to a family B polymerase, wherein thefusion protein has reverse transcriptase activity.

In certain embodiments, a method of amplifying an RNA template isprovided, wherein the method comprises subjecting a reaction mixture toa primer extension reaction, wherein the reaction mixture comprises theRNA template, at least one primer, and a fusion protein comprising anucleic acid binding polypeptide and a polymerase, wherein thepolymerase is a family B polymerase, a fragment of a family Bpolymerase, or a polypeptide having at least 80% identity to a family Bpolymerase, wherein the fusion protein has reverse transcriptaseactivity.

In certain embodiments, a method of amplifying a nucleic acid sequenceis provided, wherein the method comprises subjecting a reaction mixtureto a primer extension reaction, wherein the reaction mixture comprisesthe nucleic acid sequence, at least one primer, and a fusion proteincomprising a nucleic acid binding polypeptide and a polymerase, whereinthe reaction mixture has a pH equal to or greater than 8.5.

In certain embodiments, a fusion protein is provided, wherein the fusionprotein comprises: a polypeptide comprising an amino acid sequence of anucleic acid binding polypeptide or a fragment thereof having nucleicacid binding activity; and a reverse transcriptase.

In certain embodiments, a method of generating DNA from an RNA templateis provided, wherein the method comprises exposing the RNA template toat least one primer and a fusion protein that comprises: a polypeptidecomprising an amino acid sequence of a nucleic acid binding polypeptideor a fragment thereof having nucleic acid binding activity; and areverse transcriptase.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows agarose gel electrophoresis of two sets of reactionmixtures subjected to “fast” PCR in which the annealing temperaturesexceeded the predicted Tm of the primers, according to the workdescribed in Example D. In sets 1 and 2, lanes B, C, and D, theamplification reaction mixture included a fusion protein comprising anucleic acid binding polypeptide and a thermostable DNA polymerase. Insets 1 and 2, lanes A and E, the amplification reaction mixture includeda thermostable DNA polymerase, and did not include a fusion proteincomprising a nucleic acid binding polypeptide and a thermostable DNApolymerase. Reaction conditions are described in detail in Example D.

FIG. 2 shows agarose gel electrophoresis of gel-shift experimentsdescribed in Example K. FIG. 2A shows the results for the DNA:DNA duplexand the DNA:RNA duplex. FIG. 2B shows the results for the the DNA:DNAduplex and the RNA:RNA duplex.

FIG. 3 shows agarose gel electrophoresis of reaction mixtures subjectedto RT-PCR reactions described in Example L.

FIG. 4 shows agarose gel electrophoresis of reaction mixtures subjectedto PCR reactions described in Example M. The lanes from left to rightshow results with decreasing amount of enzyme as described in Example M.The designation Pae-Taq is for 10His-Pae3192-Taq.

FIG. 5 shows agarose gel electrophoresis of reaction mixtures subjectedto PCR reactions described in Example M. The designation AT is forAmpliTaq. The designation Pae-Taq is for 10His-Pae3192-Taq. Lanes 1 to 7had the following pH values tested as described in Example M: Lane 1; pH7.55; Lane 2; pH 7.7; Lane 3; pH 8.2; Lane 4; pH 8.6; Lane 5; pH 8.7;Lane 6; pH 9.07; and Lane 7; pH 9.3.

FIG. 6 shows the domain diagram for MMLV reverse transcriptase.

V. DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the word “a” or “an”means “at least one” unless specifically stated otherwise. In thisapplication, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including,” as well as other forms,such as “includes” and “included,” is not limiting. Also, terms such as“element” or “component” encompass both elements or componentscomprising one unit and elements or components that comprise more thanone unit unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises are hereby expressly incorporated by reference intheir entirety for any purpose. In the event that one or more of theincorporated documents defines a term that contradicts that term'sdefinition in this application, this application controls.

CERTAIN DEFINITIONS

A “nucleic acid binding polypeptide” refers to a polypeptide that has amolecular weight of about 6 to 11 kilodaltons and a predictedisoelectric point of about 9 to 11; that comprises less than or equal to4 arginine residues and less than or equal to 15 lysine residues; andthat has nucleic acid binding activity.

“Crenarchaeal nucleic acid binding polypeptide” refers to a naturallyoccurring Crenarchaeal polypeptide that has a molecular weight of about6 to 11 kilodaltons and a predicted isoelectric point of about 9 to 11;that comprises less than or equal to 4 arginine residues and less thanor equal to 15 lysine residues; that has nucleic acid binding activity;and that has an amino acid sequence that is less than 50% identical tothe amino acid sequence of Sso7d (SEQ ID NO:20). The Crenarchaeainclude, but are not limited to, members of the genus Pyrobaculum,Thermoproteus, Thermocladium, Caldivirga, Thermofilum, Staphylothermus,Ignicoccus, Aeropyrum, Pyrodictium, Pyrolobus, Sulfolobus, andMetallosphaera. See, e.g., Fitz-Gibbon et al. (2002) Proc. Nat'l Acad.Sci. USA 99:984-989.

“Nucleic acid binding activity” refers to the activity of a polypeptidein binding nucleic acid in at least one of the following two band-shiftassays. In the first assay (based on the assay of Guagliardi et al.(1997) J. Mol. Biol. 267:841-848), double-stranded nucleic acid (the452-bp HindIII-EcoRV fragment from the S. solfataricus lacs gene) islabeled with ³²P to a specific activity of at least about 2.5×10⁷ cpm/ug(or at least about 4000 cpm/fmol) using standard methods. See, e.g.,Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3^(rd)ed., Cold Spring Harbor Laboratory Press, NY) at 9.63-9.75 (describingend-labeling of nucleic acids). A reaction mixture is preparedcontaining at least about 0.5 μg of the polypeptide in about 10 μl ofbinding buffer (50 mM sodium phosphate buffer (pH 8.0), 10% glycerol, 25mM KCl, 25 mM MgCl₂). The reaction mixture is heated to 37° C. for tenminutes. About 1×10⁴ to 5×10⁴ cpm (or about 0.5-2 ng) of the labeleddouble-stranded nucleic acid is added to the reaction mixture andincubated for an additional ten minutes. The reaction mixture is loadedonto a native polyacrylamide gel in 0.5× Tris-borate buffer. Thereaction mixture is subjected to electrophoresis at room temperature.The gel is dried and subjected to autoradiography using standardmethods. Any detectable decrease in the mobility of the labeleddouble-stranded nucleic acid indicates formation of a binding complexbetween the polypeptide and the double-stranded nucleic acid. Suchnucleic acid binding activity may be quantified using standarddensitometric methods to measure the amount of radioactivity in thebinding complex relative to the total amount of radioactivity in theinitial reaction mixture.

In the second assay (based on the assay of Mai et al. (1998) J.Bacteriol. 180:2560-2563), about 0.5 μg each of negatively supercoiledcircular pBluescript KS(−) plasmid and nicked circular pBluescript KS(−)plasmid (Stratagene, La Jolla, Calif.) are mixed with a polypeptide at apolypeptide/DNA mass ratio of about ≧2.6. The mixture is incubated for10 minutes at 40° C. The mixture is subjected to 0.8% agarose gelelectrophoresis. DNA is visualized using an appropriate dye. Anydetectable decrease in the mobility of the negatively supercoiledcircular plasmid and/or nicked circular plasmid indicates formation of abinding complex between the polypeptide and the plasmid.

“Fusion protein” refers to a protein comprising two or more domainsjoined either covalently or noncovalently, wherein two or more of thedomains do not naturally occur in a single protein.

“Nucleic acid polymerase” or “polymerase” refers to any polypeptide thatcatalyzes the synthesis of a polynucleotide using an existingpolynucleotide as a template.

“Polymerase activity” refers to the activity of a nucleic acidpolymerase in catalyzing the template-directed synthesis of a newpolynucleotide. Polymerase activity is measured using the followingassay, which is based on that of Lawyer et al. (1989) J. Biol. Chem.264:6427-647. Serial dilutions of polymerase are prepared in dilutionbuffer (20 mM Tris Cl, pH 8.0, 50 mM KCl, 0.5% NP 40, and 0.5%Tween-20). For each dilution, 5 μl is removed and added to 45 μl of areaction mixture containing 25 mM TAPS (pH 9.25), 50 mM KCl, 2 mM MgCl₂,0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dTTP, 0.1 mM dCTP, 12.5 μg activatedDNA, 100 μM [α-³²P] dCTP (0.05 μCi/nmol) and sterile deionized water.The reaction mixtures are incubated at 37° C. (or 74° C. forthermostable DNA polymerases) for 10 minutes and then stopped byimmediately cooling the reaction to 4° C. and adding 10 μl of ice-cold60 mM EDTA. A 25 μl aliquot is removed from each reaction mixture.Unincorporated radioactively labeled dCTP is removed from each aliquotby gel filtration (Centri-Sep, Princeton Separations, Adelphia, N.J.).The column eluate is mixed with scintillation fluid (1 ml).Radioactivity in the column eluate is quantified with a scintillationcounter to determine the amount of product synthesized by thepolymerase. One unit of polymerase activity is defined as the amount ofpolymerase necessary to synthesize 10 nmole of product in 30 minutes.

“DNA polymerase” refers to a nucleic acid polymerase that catalyzes thesynthesis of DNA using an existing polynucleotide as a template.

“Thermostable DNA polymerase” refers to a DNA polymerase that, at atemperature higher than 37° C., retains its ability to add at least onenucleotide onto the 3′ end of a primer or primer extension product thatis annealed to a target nucleic acid sequence. In certain embodiments, athermostable DNA polymerase remains active at a temperature greater thanabout 37° C. In certain embodiments, a thermostable DNA polymeraseremains active at a temperature greater than about 42° C. In certainembodiments, a thermostable DNA polymerase remains active at atemperature greater than about 50° C. In certain embodiments, athermostable DNA polymerase remains active at a temperature greater thanabout 60° C. In certain embodiments, a thermostable DNA polymeraseremains active at a temperature greater than about 70° C. In certainembodiments, a thermostable DNA polymerase remains active at atemperature greater than about 80° C. In certain embodiments, athermostable polymerase remains active at a temperature greater thanabout 90° C.

A “cold-sensitive mutant” of a thermostable DNA polymerase refers to avariant of a thermostable DNA polymerase that exhibits substantiallyreduced activity at 25° C. to 42° C. relative to its activity at 65° C.to 72° C. In certain embodiments, activity is reduced by at least 50%,75%, or 95%.

“Reverse transcriptase” refers to a nucleic acid polymerase thatcatalyzes the synthesis of DNA using an existing RNA as a template.

“Reverse transcriptase activity” refers to the activity of a nucleicacid polymerase in catalyzing the synthesis of DNA using an existing RNAas a template.

“Thermostable reverse transcriptase” refers to a reverse transcriptasethat, at a temperature higher than 37° C., retains its ability to add atleast one nucleotide onto the 3′ end of a primer or primer extensionproduct that is annealed to a target nucleic acid sequence. In certainembodiments, a thermostable reverse transcriptase remains active at atemperature greater than about 37° C. In certain embodiments, athermostable reverse transcriptase remains active at a temperaturegreater than about 42° C. In certain embodiments, a thermostable reversetranscriptase remains active at a temperature greater than about 50° C.In certain embodiments, a thermostable reverse transcriptase remainsactive at a temperature greater than about 60° C. In certainembodiments, a thermostable reverse transcriptase remains active at atemperature greater than about 70° C. In certain embodiments, athermostable reverse transcriptase remains active at a temperaturegreater than about 80° C. In certain embodiments, a thermostablepreverse transcriptase remains active at a temperature greater thanabout 90° C.

“Processivity” refers to the extent of polymerization by a nucleic acidpolymerase during a single contact between the polymerase and itstemplate. The extent of polymerization refers to the number ofnucleotides added by the polymerase during a single contact between thepolymerase and its template.

“Percent identity” or “% identity,” with reference to nucleic acidsequences, refers to the percentage of identical nucleotides between atleast two polynucleotide sequences aligned using the Basic LocalAlignment Search Tool (BLAST) engine. See Tatusova et al. (1999) FEMSMicrobiol Lett. 174:247-250. The BLAST engine (version 2.2.10) isprovided to the public by the National Center for BiotechnologyInformation (NCBI), Bethesda, Md. To align two polynucleotide sequences,the “Blast 2 Sequences” tool is used, which employs the “blastn” programwith parameters set at default values as follows:

Matrix: not applicable

Reward for match: 1

Penalty for mismatch: −2

Open gap: 5 penalties

Extension gap: 2 penalties

Gapx dropoff: 50

Expect: 10.0

Word size: 11

Filter: on

“Percent identity” or “% identity,” with reference to polypeptidesequences, refers to the percentage of identical amino acids between atleast two polypeptide sequences aligned using the Basic Local AlignmentSearch Tool (BLAST) engine. See Tatusova et al. (1999) FEMS MicrobiolLett. 174:247-250. The BLAST engine (version 2.2.10) is provided to thepublic by the National Center for Biotechnology Information (NCBI),Bethesda, Md. To align two polypeptide sequences, the “Blast 2Sequences” tool is used, which employs the “blastp” program withparameters set at default values as follows:

Matrix: BLOSUM62

Open gap: 11 penalties

Extension gap: 1 penalty

Gap_x dropoff: 50

Expect: 10.0

Word size: 3

Filter: on

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers containing naturally occurring aminoacids as well as amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid. The amino acid polymers can be of any length.

A “fragment” of a reference polypeptide refers to a contiguous stretchof amino acids from any portion of the reference polypeptide. A fragmentmay be of any length that is less than the length of the referencepolypeptide.

A “variant” of a reference polypeptide refers to a polypeptide havingone or more amino acid substitutions, deletions, or insertions relativeto the reference polypeptide. Exemplary conservative substitutionsinclude, but are not limited to, those set forth below: TABLE 1Exemplary Amino Acid Substitutions Original Exemplary ResiduesSubstitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln Asp Glu CysSer, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg Ile Leu,Val, Met, Ala, Phe, Norleucine Leu Norleucine, Ile, Val, Met, Ala, PheLys Arg, 1,4 Diamino-butyric Acid, Gln, Asn Met Leu, Phe, Ile Phe Leu,Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, Phe TyrTrp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala, Norleucine

“Nucleic acid modification enzyme” refers to an enzymatically activepolypeptide that acts on a nucleic acid substrate. Nucleic acidmodification enzymes include, but are not limited to, nucleic acidpolymerases (such as DNA polymerases and RNA polymerases), nucleases(including endonucleases, such as restriction endonucleases, andexonucleases, such as 3′ or 5′ exonucleases), gyrases, topoisomerases,methylases, and ligases. In certain embodiments, a nucleic acidmodification enzyme is a reverse transcriptase.

“Melting temperature” or “Tm” refers to the temperature at which 50% ofthe base pairs in a double-stranded nucleic acid have denatured.“Predicted Tm” refers to the Tm calculated for a nucleic acid of >50bases in length using the following equation:Tm=81.5° C.+16.6 log₁₀ [M ⁺]+0.41(%[G+C])−675/nwhere [M⁺] is the monovalent cation concentration and n is the length ofthe nucleic acid in bases. See Rychlik et al. (1990) Nucleic Acids Res.18:6409-6412. For an oligonucleotide of ≦50 bases in length, thefollowing equation is used to calculate Tm based on nearest neighborthermodynamics:${Tm} = {\frac{\in \quad{H^{\circ} \times 1000}}{\in {S^{\circ} + {R \times {\ln\left( {C_{T}/4} \right)}}}} - 273.15 + {16.6\quad{\log_{10}\left\lbrack {M +} \right\rbrack}}}$

where εH° is the sum of the nearest neighbor enthalpy changes(kcal/mol), εS° is the sum of the nearest neighbor entropy changes(cal/K·mol), R is the molar gas constant (1.987 cal/K·mol); C_(T) is thetotal molar concentration of oligonucleotide strands; and M⁺ is themonovalent cation concentration. SantaLucia (1998) Proc. Natl Acad. Sci.USA 95:1460-1465. Values for nearest neighbor enthalpy and entropychanges are found in SantaLucia et al., supra.

The term “nucleotide base,” as used herein, refers to a substituted orunsubstituted aromatic ring or rings. In certain embodiments, thearomatic ring or rings contain at least one nitrogen atom. In certainembodiments, the nucleotide base is capable of forming Watson-Crickand/or Hoogsteen hydrogen bonds with an appropriately complementarynucleotide base. Exemplary nucleotide bases and analogs thereof include,but are not limited to, naturally occurring nucleotide bases adenine,guanine, cytosine, 6 methyl-cytosine, uracil, thymine, and analogs ofthe naturally occurring nucleotide bases, e.g., 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, N6 -Δ2-isopentenyladenine (6iA), N6 -Δ2 -isopentenyl-2-methylthioadenine(2ms6iA), N2 -dimethylguanine (dmG), 7-methylguanine (7 mG), inosine,nebularine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine,hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine,5-propynylcytosine, isocytosine, isoguanine, 7-deazaguanine,2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil,O⁶-methylguanine, N⁶-methyladenine, O⁴-methylthymine,5,6-dihydrothymine, 5,6-dihydrouracil, pyrazolo[3,4-D]pyrimidines (see,e.g., U.S. Pat. Nos. 6,143,877 and 6,127,121 and PCT publishedapplication WO 01/38584), ethenoadenine, indoles such as nitroindole and4-methylindole, and pyrroles such as nitropyrrole. Certain exemplarynucleotide bases can be found, e.g., in Fasman (1989) Practical Handbookof Biochemistry and Molecular Biology, pages 385-394, (CRC Press, BocaRaton, Fla.) and the references cited therein.

The term “nucleotide,” as used herein, refers to a compound comprising anucleotide base linked to the C-1′ carbon of a sugar, such as ribose,arabinose, xylose, and pyranose, and sugar analogs thereof. The termnucleotide also encompasses nucleotide analogs. The sugar may besubstituted or unsubstituted. Substituted ribose sugars include, but arenot limited to, those riboses in which one or more of the carbon atoms,for example the 2′-carbon atom, is substituted with one or more of thesame or different Cl, F, —R, —OR, —NR₂ or halogen groups, where each Ris independently H, C₁-C₆ alkyl or C₅-C₁₄ aryl. Exemplary ribosesinclude, but are not limited to, 2′-(C1-C6)alkoxyribose, 2′-(C5-C14)aryloxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose,2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose,2′-deoxy-3′-aminoribose, 2′-deoxy-3′-(C1-C6)alkylribose,2′-deoxy-3′-(C1-C6)alkoxyribose and 2′-deoxy-3′-(C5-C14)aryloxyribose,ribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g., 2′-O-methyl,4′-α-anomeric nucleotides, 1′-α-anomeric nucleotides, 2′-4′- and3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications(see, e.g., PCT published application nos. WO 98/22489, WO 98/39352;,and WO 99/14226). Exemplary LNA sugar analogs within a polynucleotideinclude, but are not limited to, the structures:

where B is any nucleotide base.

Modifications at the 2′- or 3′-position of ribose include, but are notlimited to, hydrogen, hydroxy, methoxy, ethoxy, allyloxy, isopropoxy,butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino,alkylamino, fluoro, chloro and bromo. Nucleotides include, but are notlimited to, the natural D optical isomer, as well as the L opticalisomer forms (see, e.g., Garbesi (1993) Nucl. Acids Res. 21:4159-65;Fujimori (1990) J. Amer. Chem. Soc. 112:7435; Urata, (1993) NucleicAcids Symposium Ser. No. 29:69-70). When the nucleotide base is purine,e.g. A or G, the ribose sugar is attached to the N⁹-position of thenucleotide base. When the nucleotide base is pyrimidine, e.g. C, T or U,the pentose sugar is attached to the N¹-position of the nucleotide base,except for pseudouridines, in which the pentose sugar is attached to theC5 position of the uracil nucleotide base (see, e.g., Kornberg andBaker, (1992) DNA Replication, 2^(nd) Ed., Freeman, San Francisco,Calif.).

One or more of the pentose carbons of a nucleotide may be substitutedwith a phosphate ester having the formula:

where α is an integer from 0 to 4. In certain embodiments, α is 2 andthe phosphate ester is attached to the 3′- or 5′-carbon of the pentose.In certain embodiments, the nucleotides are those in which thenucleotide base is a purine, a 7-deazapurine, a pyrimidine, or an analogthereof. “Nucleotide 5′-triphosphate” refers to a nucleotide with atriphosphate ester group at the 5′ position, and is sometimes denoted as“NTP”, or “dNTP” and “ddNTP” to particularly point out the structuralfeatures of the ribose sugar. The triphosphate ester group may includesulfur substitutions for the various oxygens, e.g. α-thio-nucleotide5′-triphosphates. For a review of nucleotide chemistry, see: Shabarova,Z. and Bogdanov, A. Advanced Organic Chemistry of Nucleic Acids, VCH,New York, 1994.

The term “nucleotide analog,” as used herein, refers to embodiments inwhich the pentose sugar and/or the nucleotide base and/or one or more ofthe phosphate esters of a nucleotide may be replaced with its respectiveanalog. In certain embodiments, exemplary pentose sugar analogs arethose described above. In certain embodiments, the nucleotide analogshave a nucleotide base analog as described above. In certainembodiments, exemplary phosphate ester analogs include, but are notlimited to, alkylphosphonates, methylphosphonates, phosphoramidates,phosphotriesters, phosphorothioates, phosphorodithioates,phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates,phosphoroanilidates, phosphoroamidates, boronophosphates, etc., and mayinclude associated counterions.

Also included within the definition of “nucleotide analog” arenucleotide analog monomers that can be polymerized into polynucleotideanalogs in which the DNA/RNA phosphate ester and/or sugar phosphateester backbone is replaced with a different type of internucleotidelinkage. Exemplary polynucleotide analogs include, but are not limitedto, peptide nucleic acids, in which the sugar phosphate backbone of thepolynucleotide is replaced by a peptide backbone.

As used herein, the terms “polynucleotide,” “oligonucleotide,” and“nucleic acid” are used interchangeably and mean single-stranded anddouble-stranded polymers of nucleotide monomers, including2′-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked byinternucleotide phosphodiester bond linkages, or internucleotideanalogs, and associated counter ions, e.g., H⁺, NH₄ ⁺, trialkylammonium,Mg²⁺, Na⁺ and the like. A nucleic acid may be composed entirely ofdeoxyribonucleotides, entirely of ribonucleotides, or chimeric mixturesthereof. The nucleotide monomer units may comprise any of thenucleotides described herein, including, but not limited to, naturallyoccurring nucleotides and nucleotide analogs. Nucleic acids typicallyrange in size from a few monomeric units, e.g. 5-50 when they aresometimes referred to in the art as oligonucleotides, to severalthousands of monomeric nucleotide units. Unless denoted otherwise,whenever a nucleic acid sequence is represented, it will be understoodthat the nucleotides are in 5′ to 3′ order from left to right and that“A” denotes deoxyadenosine or an analog thereof, “C” denotesdeoxycytidine or an analog thereof, “G” denotes deoxyguanosine or ananalog thereof, “T” denotes thymidine or an analog thereof, and “U”denotes uridine or an analog thereof, unless otherwise noted.

Nucleic acids include, but are not limited to, genomic DNA, cDNA, hnRNA,mRNA, rRNA, tRNA, fragmented nucleic acid, nucleic acid obtained fromsubcellular organelles such as mitochondria or chloroplasts, and nucleicacid obtained from microorganisms or DNA or RNA viruses that may bepresent on or in a biological sample. Nucleic acids include, but are notlimited to, synthetic or in vitro transcription products.

Nucleic acids may be composed of a single type of sugar moiety, e.g., asin the case of RNA and DNA, or mixtures of different sugar moieties,e.g., as in the case of RNA/DNA chimeras. In certain embodiments,nucleic acids are ribopolynucleotides and 2′-deoxyribopolynucleotidesaccording to the structural formulae below:

wherein each B is independently the base moiety of a nucleotide, e.g., apurine, a 7-deazapurine, a pyrimidine, or an analog nucleotide; each mdefines the length of the nucleic acid and can range from zero tothousands, tens of thousands, or even more; each R is independentlyselected from the group comprising hydrogen, halogen, —R″, —OR″, and—NR″R″, where each R″ is independently (C1-C6) alkyl or (C5-C14) aryl,or two adjacent Rs are taken together to form a bond such that theribose sugar is 2′,3′-didehydroribose; and each R′ is independentlyhydroxyl or

where α is zero, one or two.

In certain embodiments of the ribopolynucleotides and2′-deoxyribopolynucleotides illustrated above, the nucleotide bases Bare covalently attached to the C1′ carbon of the sugar moiety aspreviously described.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” mayalso include nucleic acid analogs, polynucleotide analogs, andoligonucleotide analogs. The terms “nucleic acid analog”,“polynucleotide analog” and “oligonucleotide analog” are usedinterchangeably and, as used herein, refer to a nucleic acid thatcontains at least one nucleotide analog and/or at least one phosphateester analog and/or at least one pentose sugar analog. Also includedwithin the definition of nucleic acid analogs are nucleic acids in whichthe phosphate ester and/or sugar phosphate ester linkages are replacedwith other types of linkages, such as N-(2-aminoethyl)-glycine amidesand other amides (see, e.g., Nielsen et al., 1991, Science254:1497-1500; WO 92/20702; U.S. Pat. No. 5,719,262; U.S. Pat. No.5,698,685;); morpholinos (see, e.g., U.S. Pat. No. 5,698,685; U.S. Pat.No. 5,378,841; U.S. Pat. No. 5,185,144); carbamates (see, e.g., Stirchak& Summerton, 1987, J. Org. Chem. 52: 4202); methylene(methylimino) (see,e.g., Vasseur et al., 1992, J. Am. Chem. Soc. 114:4006);3′-thioformacetals (see, e.g., Jones et al., 1993, J. Org. Chem. 58:2983); sulfamates (see, e.g., U.S. Pat. No. 5,470,967);2-aminoethylglycine, commonly referred to as PNA (see, e.g., Buchardt,WO 92/20702; Nielsen (1991) Science 254:1497-1500); and others (see,e.g., U.S. Pat. No. 5,817,781; Frier & Altman, 1997, Nucl. Acids Res.25:4429 and the references cited therein). Phosphate ester analogsinclude, but are not limited to, (i) C₁-C₄ alkylphosphonate, e.g.methylphosphonate; (ii) phosphoramidate; (iii) C₁-C₆alkyl-phosphotriester; (iv) phosphorothioate; and (v)phosphorodithioate.

A “target,” “target nucleic acid,” or “target nucleic acid sequence” isa nucleic acid in a sample. In certain embodiments, a target nucleicacid sequence serves as a template for amplification in a primerextension reaction, such as PCR. In certain embodiments, a targetnucleic acid sequence is an amplification product. Target nucleic acidsequences may include both naturally occurring and synthetic molecules.

In this application, a statement that one sequence is the same as or iscomplementary to another sequence encompasses situations where both ofthe sequences are completely the same or complementary to one another,and situations where only a portion of one of the sequences is the sameas, or is complementary to, a portion or the entirety of the othersequence. Here, the term “sequence” encompasses, but is not limited to,nucleic acid sequences, polynucleotides, oligonucleotides, probes, andprimers.

In this application, a statement that one sequence is complementary toanother sequence encompasses situations in which the two sequences havemismatches. Here, the term “sequence” encompasses, but is not limitedto, nucleic acid sequences, polynucleotides, oligonucleotides, probes,and primers. Despite the mismatches, the two sequences shouldselectively hybridize to one another under appropriate conditions.

The term “selectively hybridize” means that, for particular identicalsequences, a substantial portion of the particular identical sequenceshybridize to a given desired sequence or sequences, and a substantialportion of the particular identical sequences do not hybridize to otherundesired sequences. A “substantial portion of the particular identicalsequences” in each instance refers to a portion of the total number ofthe particular identical sequences, and it does not refer to a portionof an individual particular identical sequence. In certain embodiments,“a substantial portion of the particular identical sequences” means atleast 70% of the particular identical sequences. In certain embodiments,“a substantial portion of the particular identical sequences” means atleast 80% of the particular identical sequences. In certain embodiments,“a substantial portion of the particular identical sequences” means atleast 90% of the particular identical sequences. In certain embodiments,“a substantial portion of the particular identical sequences” means atleast 95% of the particular identical sequences.

In certain embodiments, the number of mismatches that may be present mayvary in view of the complexity of the composition. Thus, in certainembodiments, the more complex the composition, the more likely undesiredsequences will hybridize. For example, in certain embodiments, with agiven number of mismatches, a probe may more likely hybridize toundesired sequences in a composition with the entire genomic DNA than ina composition with fewer DNA sequences, when the same hybridization andwash conditions are employed for both compositions. Thus, that givennumber of mismatches may be appropriate for the composition with fewerDNA sequences, but fewer mismatches may be more optimal for thecomposition with the entire genomic DNA.

In certain embodiments, sequences are complementary if they have no morethan 20% mismatched nucleotides. In certain embodiments, sequences arecomplementary if they have no more than 15% mismatched nucleotides. Incertain embodiments, sequences are complementary if they have no morethan 10% mismatched nucleotides. In certain embodiments, sequences arecomplementary if they have no more than 5% mismatched nucleotides.

In this application, a statement that one sequence hybridizes or bindsto another sequence encompasses situations where the entirety of both ofthe sequences hybridize or bind to one another, and situations whereonly a portion of one or both of the sequences hybridizes or binds tothe entire other sequence or to a portion of the other sequence. Here,the term “sequence” encompasses, but is not limited to, nucleic acidsequences, polynucleotides, oligonucleotides, probes, and primers.

The term “primer” refers to a polynucleotide that anneals to a targetpolynucleotide and allows the synthesis from its 3′ end of a sequencecomplementary to the target polynucleotide.

The term “primer extension reaction” refers to a reaction in which apolymerase catalyzes the template-directed synthesis of a nucleic acidfrom the 3′ end of a primer. The term “primer extension product” refersto the resultant nucleic acid. A non-limiting exemplary primer extensionreaction is the polymerase chain reaction (PCR). The terms “extending”and “extension” refer to the template-directed synthesis of a nucleicacid from the 3′ end of a primer, which is catalyzed by a polymerase.

The term “amplifying” encompasses both linear and exponentialamplification of nucleic acid using, for example, any of a broad rangeof primer extension reactions. Exemplary primer extension reactionsinclude, but are not limited to, PCR.

The term “probe” comprises a polynucleotide that comprises a specificportion designed to hybridize in a sequence-specific manner with acomplementary region of a specific nucleic acid sequence, e.g., a targetpolynucleotide. In certain embodiments, the specific portion of theprobe may be specific for a particular sequence, or alternatively, maybe degenerate, e.g., specific for a set of sequences. In certainembodiments, a probe is capable of producing a detectable signal.

The terms “annealing” and “hybridization” are used interchangeably andmean the base-pairing interaction of one nucleic acid with anothernucleic acid that results in the formation of a duplex, triplex, orother higher-ordered structure. In certain embodiments, the primaryinteraction is base specific, e.g., A/T and G/C, by Watson/Crick andHoogsteen-type hydrogen bonding. In certain embodiments, base-stackingand hydrophobic interactions may also contribute to duplex stability.

The terms “denature” and “denaturing” refer to converting at least aportion of a double-stranded nucleic acid into nucleic acid strands thatare no longer base-paired.

The term “thermophilic microbe” refers to a microbe that grows optimallyat a temperature greater than 40° C.

The term “plurality” refers to “at least two.”

The term “label” refers to any molecule that can be detected. In certainembodiments, a label can be a moiety that produces a signal or thatinteracts with another moiety to produce a signal. In certainembodiments, a label can interact with another moiety to modify a signalof the other moiety. In certain embodiments, the signal from a labeljoined to a probe increases when the probe hybridizes to a complementarytarget nucleic acid sequence. In certain embodiments, the signal from alabel joined to a probe increases when the probe is cleaved. In certainembodiments, the signal from a label joined to a probe increases whenthe probe is cleaved by an enzyme having 5′ to 3′ exonuclease activity.

Exemplary labels include, but are not limited to, light-emitting orlight-absorbing compounds which generate or quench a detectablefluorescent, chemiluminescent, or bioluminescent signal (see, e.g.,Kricka, L. in Nonisotopic DNA Probe Techniques (1992), Academic Press,San Diego, pp. 3-28). Fluorescent reporter dyes useful as labelsinclude, but are not limited to, fluoresceins (see, e.g., U.S. Pat. Nos.5,188,934; 6,008,379; and 6,020,481), rhodamines (see, e.g., U.S. Pat.Nos. 5,366,860; 5,847,162; 5,936,087; 6,051,719; and 6,191,278),benzophenoxazines (see, e.g., U.S. Pat. No. 6,140,500), energy-transferfluorescent dyes comprising pairs of donors and acceptors (see, e.g.,U.S. Pat. Nos. 5,863,727; 5,800,996; and 5,945,526), and cyanines (see,e.g., Kubista, WO 97/45539), as well as any other fluorescent moietycapable of generating a detectable signal. Examples of fluorescein dyesinclude, but are not limited to, 6-carboxyfluorescein;2′,4′,1,4,-tetrachlorofluorescein; and2′,4′,5′,7′,1,4-hexachlorofluorescein.

Exemplary labels include, but are not limited to, quantum dots. “Quantumdots” refer to semiconductor nanocrystalline compounds capable ofemitting a second energy in response to exposure to a first energy.Typically, the energy emitted by a single quantum dot always has thesame predictable wavelength. Exemplary semiconductor nanocrystallinecompounds include, but are not limited to, crystals of CdSe, CdS, andZnS. Suitable quantum dots according to certain embodiments aredescribed, e.g., in U.S. Pat. Nos. 5,990,479 and 6,207,392 B1; Han etal. (2001) Nature Biotech. 19:631-635; and Medintz et al. (2005) Nat.Mat. 4:435-446.

Exemplary labels include, but are not limited to, phosphors andluminescent molecules. Exemplary labels include, but are not limited to,fluorophores, radioisotopes, chromogens, enzymes, antigens, heavymetals, dyes, magnetic probes, phosphorescence groups, chemiluminescentgroups, and electrochemical detection moieties. Exemplary fluorophoresinclude, but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5(Cy 5), fluorescein, Vic™, Liz™, Tamra™, 5-Fam™, 6-Fam™, and Texas Red(Molecular Probes, Eugene, Oreg.). (Vic™, Liz™, Tamra™, 5-Fam™, and6-Fam™ are all available from Applied Biosystems, Foster City, Calif.)Exemplary radioisotopes include, but are not limited to, ³²P, ³³P, and³⁵S. Exemplary labels also include elements of multi-element indirectreporter systems, e.g., biotin/avidin, antibody/antigen,ligand/receptor, enzyme/substrate, and the like, in which the elementinteracts with other elements of the system in order to effect adetectable signal. One exemplary multi-element reporter system includesa biotin reporter group attached to a primer and an avidin conjugatedwith a fluorescent label.

Exemplary detailed protocols for certain methods of attaching labels tooligonucleotides and polynucleotides can be found in, among otherplaces, Hermanson, Bioconjuqate Techniques, Academic Press, San Diego,Calif. (1996) and Beaucage et al., Current Protocols in Nucleic AcidChemistry, John Wiley & Sons, New York, N.Y. (2000). Certain exemplarynon-radioactive labeling methods, techniques, and reagents are reviewedin: Garman Non-Radioactive Labelling, A Practical Introduction, AcademicPress, San Diego (1997).

The term “indicator molecule” refers to any molecule that is capable ofproducing or effecting a detectable signal when a target nucleic acid ispresent in a sample. Exemplary indicator molecules include, but are notlimited to, SYBR® Green I, SYBR® Gold, and the like.

The term “indicator probe” refers to a probe that is capable ofproducing or effecting a detectable signal when a target nucleic acid ispresent in a sample. In certain embodiments, selective hybridization ofan indicator probe to a target nucleic acid results in the production ofa detectable signal. In certain embodiments, an indicator probe is notextendable by a polymerase. In certain embodiments, an indicator probeis extendable by a polymerase.

The term “interaction probe” refers to a probe comprising at least twomoieties that can interact with one another, wherein at least one of themoieties is capable of producing a detectable signal, and wherein thedetectable signal from the moiety increases or decreases depending uponits proximity to the other moiety. In certain embodiments employinginteraction probes, the proximity of the two moieties to one anotherdepends upon whether a target nucleic acid is present or absent in asample. In certain embodiments, the at least two moieties comprise asignal moiety and a quencher moiety. In certain embodiments, the atleast two moieties comprise a signal moiety and a donor moiety.Exemplary interaction probes include, but are not limited to, TAQMAN®probes, molecular beacons, ECLIPSE™ probes, SCORPION® primers, and thelike.

The term “5′-nuclease probe” refers to a probe that comprises a signalmoiety linked to a quencher moiety or a donor moiety through a shortoligonucleotide link element. When the 5′-nuclease probe is intact, thequencher moiety or the donor moiety influences the detectable signalfrom the signal moiety. According to certain embodiments, the5′-nuclease probe selectively hybridizes to a target nucleic acidsequence and is cleaved by a polypeptide having 5′ to 3′ exonucleaseactivity, e.g., when the probe is replaced by a newly polymerized strandduring a primer extension reaction, such as PCR.

When the oligonucleotide link element of the 5′-nuclease probe iscleaved, the detectable signal from the signal moiety changes when thesignal moiety becomes further separated from the quencher moiety or thedonor moiety. In certain embodiments that employ a quencher moiety, thedetectable signal from the signal moiety increases when the signalmoiety becomes further separated from the quencher moiety. In certainembodiments that employ a donor moiety, the detectable signal from thesignal moiety decreases when the signal moiety becomes further separatedfrom the donor moiety.

The term “hybridization-dependent probe” refers to a probe comprising asignal moiety linked to a quencher moiety or a donor moiety through anoligonucleotide link element. When the hybridization-dependent probe isnot hybridized to a target nucleic acid, the probe adopts a conformationthat allows the quencher moiety or donor moiety to come intosufficiently close proximity to the signal moiety, such that thequencher moiety or donor moiety influences a detectable signal from thesignal moiety.

The term “hairpin probe” refers to a hybridization-dependent probe thatcomprises a signal moiety linked to a quencher moiety or a donor moietythrough an oligonucleotide capable of forming a hairpin, or stem-loop,structure.

In certain embodiments of a hairpin probe, the signal moiety andquencher moiety are sufficiently close when the probe assumes a hairpinconformation, such that the quencher moiety decreases the detectablesignal from the signal moiety. When the probe is not in a hairpinconformation (e.g., when the hairpin probe is denatured or is hybridizedto a target nucleic acid sequence), the proximity of the quencher moietyand the signal moiety decreases relative to their proximity in thehairpin conformation. The decrease in proximity produces an increase inthe detectable signal from the signal moiety.

In certain embodiments of a hairpin probe, the signal moiety and donormoiety are sufficiently close when the probe assumes a hairpinconformation, such that the donor moiety increases the detectable signalfrom the signal moiety. When the probe is not in a hairpin conformation(e.g., when the hairpin probe is denatured or is hybridized to a targetnucleic acid sequence), the proximity of the donor moiety and the signalmoiety decreases relative to their proximity in the hairpinconformation. The decrease in proximity produces an decrease in thedetectable signal from the signal moiety.

The term “quencher moiety” refers to a moiety that causes the detectablesignal of a signal moiety to decrease when the quencher moiety issufficiently close to the signal moiety.

The term “donor moiety” refers to a moiety that causes the detectablesignal of a signal moiety to increase when the donor moiety issufficiently close to the signal moiety.

The term “signal moiety” refers to a moiety that is capable of producinga detectable signal.

The term “detectable signal” refers to a signal that is capable of beingdetected under certain conditions. In certain embodiments, a detectablesignal is detected when it is present in a sufficient quantity.

A. Certain Nucleic Acid Binding Polypeptides

In certain embodiments, a nucleic acid binding polypeptide comprises anaturally occurring nucleic acid binding polypeptide derived from athermophilic microbe. In certain embodiments, a nucleic acid bindingpolypeptide comprises a naturally occurring nucleic acid bindingpolypeptide derived from a hyperthermophilic archaeote. In certain suchembodiments, the hyperthermophilic archaeote is of the genus Sulfolobus.Certain small, basic nucleic acid binding polypeptides from Sulfolobussolfataricus and Sulfolobus acidocaldarius are known to those skilled inthe art. See Gao et al. (1998) Nature Struct. Biol. 5:782-786; Robinsonet al. (1998) Nature 392:202-205; McAfee et al. (1995) Biochem.34:10063-10077; and Baumann et al. (1994) Nature Struct. Biol.1:808-819. Certain such polypeptides include, but are not limited to,Sso7d and Sac7d, which bind DNA in a sequence non-specific manner. SeeGao et al. (1998) Nature Struct. Biol. 5:782-786; Robinson et al. (1998)Nature 392:202-205; McAfee et al. (1995) Biochem. 34:10063-10077; andBaumann et al. (1994) Nature Struct. Biol. 1:808-819.

Sso7d and Sac7d are of relatively low molecular weight (about 7 kDa) andare rich in lysine residues. Id. Certain lysine residues are believed tobe involved in DNA binding. See Gao et al. (1998) Nature Struct. Biol.5:782-786. Both protect double-stranded DNA from thermal denaturation byincreasing its melting temperature (Tm) by about 40° C. Id.; Robinson etal. (1998) Nature 392:202-205. Sso7d also promotes the annealing ofcomplementary DNA strands at temperatures exceeding the predicted Tm ofthe resulting duplex. See Guagliardi et al. (1997) J. Mol. Biol.267:841-848. Sso7d exhibits a strong preference for DNA strands that arecomplementary without any mismatches over DNA strands that contain evena single mismatch. See id.; U.S. Patent Application Publication No. US2003/0022162 A1. It is postulated that small, basic polypeptides such asSso7d. and Sac7d protect the DNA of hyperthermophiles from denaturationand degradation in the hyperthermophilic environment, where temperaturesapproach or exceed 100° C. See Guagliardi et al. (1997) J. Mol. Biol.267:841-848.

In certain embodiments, a nucleic acid binding polypeptide comprises theamino acid sequence of Sso7d (SEQ ID NO:20). Sso7d is encoded by SEQ IDNOs:44 and 45. Sso7d is 64 amino acids in length with a predictedisolectric point of 10.2. A exemplary variant of Sso7d having fouradditional amino acids at its N-terminus is shown in SEQ ID NO:21. Thatvariant is encoded by SEQ ID NO:46.

In certain embodiments, a nucleic acid binding polypeptide comprises aCrenarchaeal nucleic acid binding polypeptide. In certain embodiments, aCrenarchaeal nucleic acid binding polypeptide comprises a naturallyoccurring polypeptide from the crenarchaeon Pyrobaculum aerophilum. Incertain embodiments, a Crenarchaeal nucleic acid binding polypeptidecomprises the amino acid sequence of Pae3192 (SEQ ID NO:1), which can befound at GenBank accession numbers ML64739 and AAL64814. Pae3192 isencoded by the open reading frames “PAE3192” (SEQ ID NO:2) and “PAE3289”(SEQ ID NO:3), which are unannotated open reading frames identified inthe complete genome sequence of P. aerophilum. See GenBank accession no.AE009441.

In certain embodiments, a Crenarchaeal nucleic acid binding polypeptidecomprises the amino acid sequence of Pae0384 (SEQ ID NO:4), which can befound at GenBank accession number ML62754. Pae0384 is encoded by theopen reading frame “PAE0384” (SEQ ID NO:5), which is an unannotated openreading frame identified in the complete genome sequence of P.aerophilum. See GenBank accession no. AE009441.

SEQ ID-NOs:1 and 4 are low molecular weight, basic proteins of 57 and 56amino acids in length, respectively, with a predicted isoelectric pointof about 10.5. SEQ ID NO:1 contains 12 lysine residues and 2 arginineresidues. SEQ ID NO:4 contains 11 lysine residues and 2 arginineresidues. SEQ ID NOs:1 and 4 are about 97% identical to each other. SEQID NOs:1 and 4 are similar in size and charge to Sso7d, but they are notsignificantly identical to the amino acid sequence of Sso7d.

Additionally, SEQ ID NO:1 contains a “KKQK” motif near its N-terminus(residues 3 to 6 of SEQ ID NO:1). This motif resembles the “KQKK” motiffound at the C-terminus of Sso7d (residues 61-64 of SEQ ID NO:20). Thelocation of these motifs at opposite termini of SEQ ID NO:1 and Sso7dmay have resulted from gene rearrangements during the divergence of thedifferent Crenarchaeal species. The KQKK motif of Sso7d is discussed inShehi et al. (2003) Biochem. 42:8362-8368.

In certain embodiments, a Crenarchaeal nucleic acid binding polypeptidecomprises a naturally occurring polypeptide from the crenarchaeonAeropyrum pernix. In certain embodiments, a Crenarchaeal nucleic acidbinding polypeptide comprises the amino acid sequence of Ape3192 (SEQ IDNO:6). SEQ ID NO:6 is 55 amino acids in length with a predictedisoelectric point of about 10.5. It contains 13 lysine residues and 3arginine residues. SEQ ID NO:6 is similar in size and charge to Sso7d,but it is not significantly identical to the amino acid sequence ofSso7d.

In certain embodiments, a nucleic acid binding polypeptide comprises afragment of a naturally occurring nucleic acid binding polypeptide. Incertain such embodiments, the fragment has at least one activity of thenaturally occurring nucleic acid binding polypeptide. Exemplaryactivities of a naturally occurring nucleic acid binding polypeptideinclude, but are not limited to, the ability to bind nucleic acid,stabilize nucleic acid duplexes from thermal denaturation, increase theTm of primers, and increase the processivity of a polymerase. Otherexemplary activities of a naturally occurring nucleic acid bindingpolypeptide include, but are not limited to the ability to promoteannealing of complementary nucleic acid strands, stabilize nuceic acidduplexes, and enhance the activity of a nucleic acid modificationenzyme. In certain embodiments, the fragment has a predicted isoelectricpoint of about 9-11.

In certain embodiments, a nucleic acid binding polypeptide comprises afragment of a polypeptide comprising an amino acid sequence selectedfrom SEQ ID NOs:1, 4, 6, 20, and 21. In certain such embodiments, thefragment lacks N-terminal amino acids. In certain such embodiments, thefragment lacks up to the first 12 N-terminal amino acids of an aminoacid sequence selected from SEQ ID NOs:1, 4, 6, 20, and 21. In certainembodiments, the fragment lacks C-terminal amino acids. In certain suchembodiments, the fragment lacks up to the last 12 C-terminal amino acidsof an amino acid sequence selected from SEQ ID NOs:1, 4, 6,20, and 21.

In certain embodiments, a nucleic acid binding polypeptide comprises avariant of a naturally occurring nucleic acid binding polypeptide. Incertain such embodiments, the variant has at least one activity of anaturally occurring nucleic acid binding polypeptide.

In certain embodiments, a nucleic acid binding polypeptide comprises avariant of a polypeptide comprising an amino acid sequence selected fromSEQ ID NOs:1, 4, 6, 20, and 21. In certain such embodiments, the variantcomprises an amino acid sequence having from about 60% to about 99%identity to an amino acid sequence selected from SEQ ID NOs:1, 4, 6, 20,and 21. For example, in certain embodiments, the variant comprises anamino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, or 99% identity to an amino acid sequence selected fromSEQ ID NOs:1, 4, 6, 20, and 21. In certain such embodiments, lysine andarginine residues are not substituted or deleted in the variant.

In certain embodiments, a variant of a Crenarchaeal nucleic acid bindingpolypeptide is provided. In certain embodiments, one or more amino acidsthat are not conserved among Crenarchaeal nucleic acid bindingpolypeptides may be substituted or deleted to create a suitable variant.For example, the first of the two alignments below demonstrates that SEQID NOs:1 and 6 have 60% identity and 74% similarity as determined by the“Blast 2 Sequence” blastp program set at default parameters. (Incalculating percent similarity, the blastp program includes bothidentical and similar amino acids. Similar amino acids are indicated by“+” signs in the alignments below.) The second of the two alignmentsbelow demonstrates that SEQ ID NOs:4 and 6 have 59% identity and 72%similarity as determined by the “Blast 2 Sequence” blastp program set atdefault parameters. In certain embodiments, one or more amino acids thatare not conserved in at least one of the alignments below (i.e., aminoacids that are not identical or similar) are substituted or deleted tocreate variants of polypeptides comprising SEQ ID NO:1, SEQ ID NO:4, orSEQ ID NO:6. SEQ ID NO:1: 1MSKKQKLKFYDIKAKQAFETDQYEVIEKQTARGPMMFAVAKSPYTGIKVYRLLGKKK 57   MKK+K+KF+D+ AK+ +ETD YEV  K+T RG   FA AKSPYTG   YR+LGK SEQ ID NO:6: 1MPKKEKIKFFDLVAKKYYETDNYEVEIKETKRGKFRFAKAKSPYTGKIKYRVLGKA 55 SEQ ID NO:4:1 MAKQKLKFYDIKAKQSFETDKYEVIEKETARGPMLFAVATSPYTGIKVYRLLGKKK 56    K+K+KF+D+ AK+ +ETD YEV  KET RG   FA A SPYTG   YR+LGK SEQ ID NO:6: 1MPKKEKIKFFDLVAKKYYETDNYEVEIKETKRGKFRFAKAKSPYTGKIFYRVLGKA 55

Based on the above alignments, a consensus sequence for a Crenarchaealnucleic acid binding polypeptide is provided as follows: SEQ ID NO:28 5′KXKXKFXDXXAKXXXETDXYEVXXKXTXRGXXXFAXAKSPYTGXXXR XLGK 3′In the above consensus sequence, “X” is any amino acid. In certainembodiments, a nucleic acid binding polypeptide comprises an amino acidsequence that conforms to that consensus sequence. In certain suchembodiments, the nucleic acid binding polypeptide has at least oneactivity of a naturally occurring nucleic acid binding polypeptide.

In certain embodiments, a fragment or variant of a naturally occurringnucleic acid binding polypeptide has nucleic acid binding activity thatis less than that of the naturally occurring nucleic acid bindingpolypeptide. In certain such embodiments, the fragment or variant hasfrom about 10-20%, about 20-30%, about 30-40%, about 40-50%, about50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-95% of thenucleic acid binding activity of the naturally occurring nucleic acidbinding polypeptide.

In certain embodiments, a polynucleotide comprising a nucleic acidsequence encoding any of the above nucleic acid binding polypeptides isprovided. In certain embodiments, a polynucleotide comprises a nucleicacid sequence encoding a polypeptide comprising an amino acid sequenceselected from SEQ ID NOs:1, 4, 6, 20, and 21. In certain embodiments, apolynucleotide comprises a nucleic acid sequence encoding a fragment ofa polypeptide comprising an amino acid sequence selected from SEQ IDNOs: 1, 4, 6, 20, and 21. In certain such embodiments, the fragment hasat least one activity of a naturally occurring nucleic acid bindingpolypeptide. In certain embodiments, a polynucleotide comprises anucleic acid sequence encoding a variant of a polypeptide comprising anamino acid sequence selected from SEQ ID NOs:1, 4, 6, 20, and 21. Incertain such embodiments, the variant has at least one activity of anaturally occurring nucleic acid binding polypeptide.

In certain embodiments, a polynucleotide comprises a nucleic acidsequence selected from SEQ ID NOs:2, 3, 5, 7, 44, 45, and 46. In certainembodiments, a polynucleotide comprises a fragment of a nucleic acidsequence selected from SEQ ID NOs: 2, 3, 5, 7, 44, 45, and 46, whereinthe fragment encodes a polypeptide having at least one activity of anaturally occurring nucleic acid binding polypeptide.

In certain embodiments, a polynucleotide comprises a variant of anucleic acid sequence selected from SEQ ID NOs:2, 3, 5, 7, 44, 45, and46, wherein the variant encodes a polypeptide having at least oneactivity of a naturally occurring nucleic acid binding polypeptide. Incertain embodiments, a variant of a nucleic acid sequence selected fromSEQ ID NOs:2, 3, 5, 7, 44, 45, and 46 comprises a nucleic acid sequencehaving from about 60% to about 99% identity to a nucleic acid sequenceselected from SEQ ID NOs:2, 3, 5, 7, 44, 45, and 46. For example, incertain embodiments, a variant of a nucleic acid sequence selected fromSEQ ID NOs:2, 3, 5, 7, 44, 45, and 46 comprises a nucleic acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identity to a nucleic acid sequence selected from SEQ ID NO:2, 3, 5, 7,44, 45, and 46. In certain such embodiments, the variant encodes apolypeptide having at least one activity of a naturally occurringnucleic acid binding polypeptide.

In certain embodiments, the length of an isolated polynucleotide is anynumber of nucleotides less than or equal to 10,000. For example, incertain embodiments, an isolated polynucleotide is less than or equal to10,000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000,1000, or 500nucleotides in length. In certain embodiments, the length of an isolatedpolynucleotide does not include vector sequences.

In certain embodiments, a polynucleotide encoding a nucleic acid bindingpolypeptide is obtained by the polymerase chain reaction (PCR). Certainmethods employing PCR are known to those skilled in the art. See, e.g.,Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Chapter 8(3^(rd) ed., Cold Spring Harbor Laboratory Press, NY). In certainembodiments, a polynucleotide comprising all or a portion of the codingsequence of a nucleic acid binding polypeptide is amplified usingappropriate primers. In certain embodiments, restriction enzyme sitesare included in the primers to facilitate cloning of the amplificationproduct into an appropriate expression vector. In certain embodiments,the polynucleotide is amplified from genomic DNA or from cDNA of acrenarchaeote. The complete genome sequence of certain crenarchaeotes ispublished and may be used in designing primers for PCR. See, e.g.,Fitz-Gibbon et al. (2002) Proc. Nat'l Acad. Sci. USA 99:984-989;Kawarabayasi (1999) DNA Research Supp:145-152; and She et al. (2001)Proc. Nat'l Acad. Sci. USA 98:7835-7840.

In certain embodiments, a polynucleotide encoding a nucleic acid bindingpolypeptide is obtained by synthesizing individual oligonucleotideswhich are ligated end-to-end in vitro, with the resulting ligationproduct comprising the coding sequence of a nucleic acid bindingpolypeptide. In certain embodiments, the ligation product is amplifiedby PCR. In certain embodiments, the oligonucleotides overlap in sequenceand are extended by PCR, resulting in a PCR product comprising thecoding sequence of a nucleic acid binding polypeptide. See, e.g.,Stemmer et al. (1995) Gene 164:49-53; Gronlund et al. (2003) J. Biol.Chem. 278:40144-40151. In certain embodiments, the PCR product is clonedinto a suitable vector.

In certain embodiments, a polynucleotide encoding a nucleic acid bindingpolypeptide is cloned into a suitable vector. In certain suchembodiments, the vector is transferred (e.g., transformed ortransfected) into a host cell. In certain embodiments, a polynucleotideencoding a nucleic acid binding polypeptide is cloned into an expressionvector and, in certain embodiments, expressed in a suitable host cell.Certain exemplary expression vectors are available for use in certainhost cells including, but not limited to, prokaryotes, yeast cells,insect cells, plant cells, and mammalian cells. See, e.g., Ausubel etal. (1991) Current Protocols in Molecular Biology, Chapter 16, JohnWiley & Sons, New York. Certain expression vectors for the inducibleexpression of recombinant proteins in prokaryotes are known to thoseskilled in the art. For example, in certain embodiments, apolynucleotide encoding a nucleic acid binding polypeptide is clonedinto an expression vector such that its transcription is under thecontrol of an inducible promoter, such as the T7 bacteriophage promoter,the T5 promoter, or the tac promoter. See, e.g., the pET series ofvectors (Invitrogen, Carlsbad, Calif.), the pQE series of vectors(Qiagen, Valencia, Calif.), or the PGEX series of vectors (AmershamBiosciences, Piscataway, N.J.). In certain embodiments, the recombinantexpression vector is transformed into bacteria, such as E. coli. Incertain embodiments, the expression of the nucleic acid bindingpolypeptide is induced by culturing the bacteria under certain growthconditions. For example, in certain embodiments, expression of thenucleic acid binding polypeptide is induced by addition ofisopropylthio-β-galactoside (IPTG) to the culture medium.

In various embodiments of expression vectors, a polynucleotide encodinga tag, such as an affinity tag, is expressed in frame with apolynucleotide encoding a nucleic acid binding polypeptide. In certainembodiments, certain such tags can provide a mechanism for detection orpurification of the nucleic acid binding polypeptide. Examples of tagsinclude, but are not limited to, polyhistidine tags, which allowpurification using nickel chelating resin, and glutathione S-transferasemoieties, which allow purification using glutathione-basedchromatography. In certain embodiments, an expression vector furtherprovides a cleavage site between the tag and the nucleic acid bindingpolypeptide, so that the nucleic acid binding polypeptide may be cleavedfrom the tag following purification. In certain embodiments, e.g.,embodiments using polyhistidine tags, the nucleic acid bindingpolypeptide is not cleaved from the tag. It has been reported that thepresence of a polyhistidine tag on a recombinant DNA binding protein mayenhance the interaction of the DNA binding protein with DNA. See, e.g.,Buning et al. (1996) Anal. Biochem. 234:227-230.

B. Certain DNA Polymerases

Certain polymerases are known to those skilled in the art. For example,DNA polymerases include DNA-dependent polymerases, which use DNA as atemplate, or RNA-dependent polymerases, such as reverse transcriptase,which use RNA as a template. Currently, DNA-dependent DNA polymerasesfall into one of six families (A, B, C, D, X, and Y), with most fallinginto one of three families (A, B, and C). See, e.g., Ito et al. (1991)Nucleic Acids Res. 19:4045-4057; Braithwaite et al. (1993) Nucleic AcidsRes. 21:787-802; Filee et al. (2002) J. Mol. Evol. 54:763-773; and Alba(2001) Genome Biol. 2:3002.1-3002.4. Certain DNA polymerases may besingle-chain polypeptides (e.g., certain family A and B polymerases) ormulti-subunit enzymes (e.g., certain family C polymerases) with one ofthe subunits having polymerase activity. Id. In certain embodiments, afusion protein comprises a DNA polymerase selected from a family A, B,C, D, X, or Y polymerase.

In certain embodiments, a polymerase comprises a fragment or variant ofan A, B, C, D, X, or Y polymerase having polymerase activity. In certainembodiments, a polymerase comprises a family A DNA polymerase or afragment or variant thereof having polymerase activity. In certain suchembodiments, the family A polymerase is a bacterial family A polymerase,such as a polymerase from the genus Bacillus, Thermus, Rhodothermus orThermotoga. In certain such embodiments, the family A polymerase is TaqDNA polymerase (SEQ ID NO:31) or a fragment or variant thereof havingpolymerase activity. In certain embodiments, a variant of Taq DNApolymerase comprises an amino acid sequence having at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:31.

In certain embodiments, a polymerase comprises a family B DNA polymeraseor a fragment or variant thereof having polymerase activity. In certainsuch embodiments, the family B polymerase is an archaeal family Bpolymerase, such as a polymerase from the genus Thermococcus,Pyrococcus, or Pyrobaculum. In certain such embodiments, the family Bpolymerase is Pfu DNA polymerase (SEQ ID NO:30) or a fragment or variantthereof having polymerase activity. In certain embodiments, a variant ofPfu DNA polymerase comprises an amino acid sequence having at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:30.

In addition to polymerase activity, certain DNA polymerases also possessother activities, such as 3′ to 5′ exonuclease (proofreading) activityor 5′ to 3′ exonuclease activity. See, e.g., Fileé et al. (2002) J. Mol.Evol. 54:763-773; and Pavlov et al. (2004) Trends in Biotech.22:253-260. In certain such DNA polymerases, polymerase activity andexonuclease activity are carried out by separate domains. The domainstructure of certain DNA polymerases is known to those skilled in theart. See, e.g., id.; Albá (2001) Genome Biol. 2:3002.1-3002.4; andSteitz (1999) J. Biol. Chem. 274:17395-17398.

In certain embodiments, a “chimeric” DNA polymerase is provided. Incertain such embodiments, a chimeric DNA polymerase comprises a domainhaving polymerase activity from a particular DNA polymerase and a domainhaving exonuclease activity from a different DNA polymerase. See, e.g.,U.S. Pat. Nos. 5,795,762 and 5,466,591.

In certain embodiments, a DNA polymerase having both polymerase activityand exonuclease activity is provided. In certain such embodiments, theexonuclease activity is 5′ to 3′ exonuclease activity. In certain suchembodiments, the level of 5′ to 3′ exonuclease activity is reduced oreliminated relative to the level of 5′ to 3′ exonuclease activity of anative DNA polymerase. In certain such embodiments, mutation of a DNApolymerase results in reduction or elimination of 5′ to 3′ exonucleaseactivity. In certain such embodiments, one or more amino acidsubstitutions result in reduction or elimination of 5′ to 3′ exonucleaseactivity. Certain such substitutions are known to those skilled in theart. For example, substitution of a conserved glycine in certainthermostable DNA polymerases reduces or eliminates 5′ to 3′ exonucleaseactivity. See, e.g., U.S. Pat. Nos. 5,795,762 and 5,466,591 (describingthe G46D substitution in Taq, Tth, and TZ05 DNA polymerases; the G43Dsubstitution in Tsps17 DNA polymerase; and the G37D substitution in Tmaand Taf DNA polymerases).

In certain embodiments, deletion of one or more amino acids from a DNApolymerase results in the reduction or elimination of 5′ to 3′exonuclease activity. Certain such deletions are known to those skilledin the art. For example, certain N-terminal deletions of certainthermostable DNA polymerases reduce or eliminate 5′ to 3′ exonucleaseactivity. Exemplary N-terminal deletions include, but are not limitedto, deletion of about the first 35-50 amino acid residues of athermostable DNA polymerase. See, e.g., U.S. Pat. Nos. 5,795,762 and5,466,591 (describing deletion of N-terminal amino acid residues up toand including the conserved glycine residues in Taq, Tth, TZ05, Tsps17,Tma, and Taf, described above). Exemplary N-terminal deletions furtherinclude, but are not limited to, deletion of about the first 70-80 aminoacid residues of a thermostable DNA polymerase. See, e.g., U.S. Pat.Nos. 5,795,762 and 5,466,591 (describing deletion of N-terminal aminoacid residues up to and including the following residues: Ala 77 (TaqDNA polymerase), Ala 78 (Tth DNA polymerase), Ala 78 (TZ05 DNApolymerase), Ala 74 (TSPS17 DNA polymerase), Leu 72 (Tma DNApolymerase), and Ile 73 (Taf DNA polymerase)). Exemplary N-terminaldeletions further include, but are not limited to, deletion of the first139 or the first 283 amino acid residues of Tma DNA polymerase. See,e.g., U.S. Pat. Nos. 5,795,762 and 5,466,591.

In certain embodiments, a DNA polymerase that lacks an exonucleasedomain is provided. In certain embodiments, the exonuclease domain is a5′ to 3′ exonuclease domain. Exemplary polymerases that lack a 5′ to 3′exonuclease domain include, but are not limited to, a family Bpolymerase such as Pfu DNA polymerase; the large “Klenow” fragment of E.coli DNA polymerase I; the “Klentaq235” fragment of Taq DNA polymerase,which lacks the first 235 N-terminal amino acids of full-length Taq; the“Klentaq278” fragment of Taq DNA polymerase, which lacks the first 278N-terminal amino acids of full-length Taq; and the “Stoffel” fragment ofTaq DNA polymerase, which lacks about the first 289-300 N-terminal aminoacids of full-length Taq DNA polymerase. See Lawyer et al. (1989) J.Biol. Chem. 264:6427-6437 (describing a “Stoffel” fragment); Vainshteinet al. (1996) Protein Science 5:1785-1792; Barnes (1992) Gene 112:29-35;and U.S. Pat. No. 5,436,149. In certain embodiments, thermostable DNApolymerases that lack a 5′ to 3′ exonuclease domain show increasedthermal stability and/or fidelity relative to their full-lengthcounterparts. See, e.g., Barnes (1992) Gene 112:29-35; and U.S. Pat. No.5,436,149.

In certain embodiments, mutation of one or more amino acids in a DNApolymerase results in the reduction or elimination of 3′ to 5′exonuclease activity. For example, the 3′ to 5′ exonuclease domain ofcertain archaeal family B polymerases comprises the consensus sequenceFDXE(TN) (where “X” is any amino acid). See, e.g., amino acid residues140-144 of SEQ ID NO:30; and Kahler et al. (2000) J. Bacteriol.182:655-663. In certain embodiments, mutation of the consensus sequenceto FDXD(T/V) reduces the level of 3′ to 5′ exonuclease activity to about10% or less of the activity in the corresponding wild-type polymerase.See, e.g., Southworth et al. (1996) Proc. Natl. Acad. Sci. USA93:5281-5285 (describing a mutant of Thermococcus sp. 9°N-7); andDerbyshire et al. (1995) Methods Enzymol. 262:363-388. In certainembodiments, mutation of the consensus sequence to FAXA(T/V)substantially eliminates 3′ to 5′ exonuclease activity. See, e.g.,Southworth et al. (1996) Proc. Natl. Acad. Sci. USA 93:5281-5285(describing a mutant of Thermococcus sp. 9°N-7); Kong et al. (1993) J.Biol. Chem. 268:1965-1975 (describing a mutant of Tli DNA polymerase);and Derbyshire et al. (1995) Methods Enzymol. 262:363-388. In certainembodiments, reducing or eliminating 3′ to 5′ exonuclease activity mayalleviate polymerase “stutter” or slippage, e.g., in the amplificationof repetitive DNA. See, e.g., Walsh et al. (1996) Nucleic Acids Res.24:2807-2812. In certain embodiments, reducing or eliminating 3′ to 5′exonuclease activity may alleviate primer degradation by the polymerase.

In certain embodiments, a DNA polymerase is provided that comprises oneor more mutations adjacent to the exonuclease domain. For example, incertain embodiments, a B family DNA polymerase from a hyperthermophilicArchaeon, such as KOD polymerase from Thermococcus kodakarensis, isprovided in which the histidine at position 147 (proximal to theconserved Exo-I domain) is changed to glutamic acid (H147E), whichresults a lowered 3′→5′ exonuclease activity while maintaining nearwild-type fidelity. The resulting measured ratio of exonuclease activityto polymerase activity is lowered, resulting in higher yields ofamplified DNA target from a typical PCR reaction. See, for example,Kuroita et al., J. Mol. Biol., 351:291-298 (2005).

In certain embodiments, a DNA polymerase is provided that comprises oneor more mutations such that it retains double stranded exonucleaseactivity, but it has reduced single stranded exonuclease activity. Anonlimiting example is a polymerase with the Y384F mutation (mutation oftyrosine to phenylalanine) in the conserved YxGG domain of family B DNApolymerases. See, for example, Bohike et al., Nucl. Acid Res.,28:3910-3917 (2000).

In certain embodiments, a family B DNA polymerase is provided thatcomprises one or more mutations that allow the polymerase to perform DNApolymerization using a primed RNA template. Exemplary polymerasesinclude, but are not limited to, a family B polymerase, such as Pfu DNApolymerase, with a point mutation L408Y or L408F (leucine to tyrosine orto phenylalane) in the conserved LYP motif, which results in apolymerase that can perform an RNA-templated DNA polymerizationreaction. See, for example, U.S. Patent Publication No. US2003/0228616.Exemplary family B polymerases include, but are not limited to, Pfupolymerase, Tgo polymerase (Roche), Vent polymerase (New EnglandBiolabs), Deep Vent polymerase (New England Biolabs), KOD polymerase(Toyo Boseki/EMD Biosciences), and 9°Nm polymerase (New EnglandBiolabs).

In certain embodiments, a DNA polymerase is provided that comprises oneor more mutations that reduce the ability of the polymerase todiscriminate against the incorporation of dideoxynucleotides. Certainexemplary mutations are described, for example, in U.S. Pat. No.6,333,183; EP 0 745 676 B1; and U.S. Pat. No. 5,614,365. One suchexemplary mutation is the F667Y mutation in Taq DNA polymerase. See,e.g., U.S. Pat. No. 5,614,365.

In certain embodiments, a DNA polymerase is provided that comprises oneor more mutations that reduce the ability of the polymerase todiscriminate against the incorporation of fluorescently labelednucleotides into polynucleotides. In certain embodiments, such“discrimination reduction” mutations occur within the nucleotide labelinteraction region of a DNA polymerase, which is described, for example,in U.S. Pat. No. 6,265,193. Exemplary discrimination reduction mutationsare provided in U.S. Pat. No. 6,265,193.

In certain embodiments, a DNA polymerase further comprises one or moremutations in addition to one or more discrimination reduction mutations.Certain exemplary mutations include, but are not limited to, mutationsthat increase or decrease 3′ to 5′ exonuclease activity; increase ordecrease 5′ to 3′ exonuclease activity; increase or decreasethermostability; increase or decrease processivity; and increaseincorporation of dideoxynucleotides. In certain embodiments, a DNApolymerase comprises one or more discrimination reduction mutations andone or more mutations that decrease 3′ to 5′ exonuclease activity. Incertain embodiments, a DNA polymerase comprises one or morediscrimination reduction mutations and one or more mutations thatincrease incorporation of dideoxynucleotides. Certain such DNApolymerases are described, for example, in U.S. Pat. No. 6,265,193.

In certain embodiments, a polymerase comprises a thermostable DNApolymerase. In certain embodiments, a thermostable DNA polymerase is anaturally occurring thermostable DNA polymerase. In certain embodiments,a thermostable DNA polymerase is a fragment or variant of a naturallyoccurring thermostable DNA polymerase that possesses polymeraseactivity. Exemplary guidance for determining certain such fragments andvariants is provided in Pavlov et al. (2004) Trends in Biotech.22:253-260.

Certain exemplary thermostable DNA polymerases are known to thoseskilled in the art. See, e.g., Sambrook et al. (2001) Molecular Cloning:A Laboratory Manual (3^(rd) ed., Cold Spring Harbor Laboratory Press,NY) at 8.10-8.1 1. Certain exemplary thermostable DNA polymerasesinclude, but are not limited to, DNA polymerases from the genus Thermus,Thermococcus, Thermotoga, Bacillus, and Pyrococcus. Certain exemplarythermostable DNA polymerases include, but are not limited to, DNApolymerases from Thermus aquaticus (e.g., Taq DNA polymerase), Thermusbrockianus (e.g., Tbr polymerase), Thermus flavus (e.g., Tfl DNApolymerase), Thermus caldophilus, Thermus filiformis, Thermus oshimai,Thermus thermophilus (e.g., Tth DNA polymerase), and Thermus ubiquitus.Certain other thermostable DNA polymerases from Thermus include, but arenot limited to, Tsps17 and TZ05. Certain fragments and variants of Taq,Tfl, Tth, Tsps17, and TZ05 DNA polymerases are known to those skilled inthe art. See, e.g., Vainshtein et al. (1996) Protein Science 5:1785-1792(discussing the Taq Stoffel fragment), EP 0 745 676 B1, WO 01/14568, US2004/0005573 A1, U.S. Pat. No. 5,795,762, and U.S. Pat. No. 5,466,591.

In certain embodiments, a polymerase comprises a variant of a naturallyoccurring thermostable DNA polymerase having increased efficiencyrelative to the naturally occurring thermostable DNA polymerase. Certainsuch variants of Taq DNA polymerase are known to those skilled in theart. One such exemplary variant is the S543N mutant of Klentaq. Thatvariant synthesizes long DNA molecules with greater efficiency thanKlentaq. See, e.g., Ignatov et al. (1999) FEBS Letters 425:249-250. Italso more efficiently amplifies templates having complex secondarystructures (e.g., GC-rich templates) that typically induce polymerasepausing. See, e.g., lgnatov et al. FEBS Letters 448:145-148.

In certain embodiments, a polymerase comprises a thermostable DNApolymerase from Thermococcus litoralis (e.g., Tli polymerase),Thermococcus kodakarensis KODI (e.g., KOD DNA polymerase), orThermococcus gorgonarius (e.g., Tgo DNA polymerase). See, e.g., Takagiet al. (1997) Appl. Environ. Microbiol. 63:4504-4510 (KOD DNApolymerase). Certain fragments and variants of KOD DNA polymerase areknown to those skilled in the art. See, e.g., EP 1 154 017 A1 and U.S.Pat. No. 5,436,149. Certain such variants having increased processivityand elongation rates are commercially available from EMDBiosciences—Novagen, San Diego, Calif. In certain embodiments, athermostable DNA polymerase comprises a DNA polymerase from Thermotoganeapolitana (e.g., Tne DNA polymerase) or Thermotoga maritima (e.g., TmaDNA polymerase). See, e.g., US 2003/0092018 A1 and US 2003/0162201 A1.In certain embodiments, a thermostable DNA polymerase comprises a DNApolymerase from Thermosipho africanus (e.g., Taf DNA polymerase).Certain fragments and variants of Tma, Taf, and Tne DNA polymerases areknown to those skilled in the art. See, e.g., US 2003/0092018 Al, US2003/0162201 A1, U.S. Pat. No. 5,795,762, and and U.S. Pat. No.5,466,591.

Certain exemplary thermostable DNA polymerases include, but are notlimited to, DNA polymerases from Pyrococcus furiosus (e.g., Pfu DNApolymerase), Pyrococcus woesei (e.g., Pwo polymerase), Pyrococcus spp.GB-D, Pyrococcus abyssi, and Pyrolobus fumarius. See, e.g., U.S. Pat.No. 5,834,285, U.S. Pat. No. 6,489,150 B1, U.S. Pat. No. 6,673,585 B1,U.S. Pat. No. 5,948,666, U.S. Pat. No. 6,492,511, and EP 0 547 359 B1.

Certain fragments and variants of Pfu polymerase are known to thoseskilled in the art. See, e.g., U.S. Pat. No. 6,333,183 B1 and US2004/0219558 A1. In certain embodiments, a variant of Pfu polymerasecomprises any of the variants described in US 2004/0219558 A1. Incertain embodiments, a variant of Pfu polymerase comprises any one ormore of the following mutations: M247R, T265R, K502R, A408S, K485R, andΔL381 (deletion).

Certain variants of Pyrococcus spp. GB-D polymerase are known to thoseskilled in the art. See, e.g., US 2004/0219558 A1. In certainembodiments, a variant of Pyrococcus spp. GB-D polymerase comprises anyof the variants described in US 2004/0219558 A1.

In certain embodiments, a variant of a Pyrococcus polymerase has one ormore mutations in the uracil binding pocket. Certain such polymerasesare capable of utilizing uracil containing templates. For example, incertain embodiments, a variant of Pfu DNA polymerase comprises the V93Qmutation. See, e.g., Shuttleworth et al. (2004) J. Molec. Biol.337:621-634; and Fogg et al. (2002) Nature Struct. Biol. 9:922-927.

In certain embodiments, a thermostable DNA polymerase comprises a DNApolymerase from Bacillus stearothermophilus or a variant or fragmentthereof, such as the “large fragment” of Bst DNA polymerase. In certainembodiments, a thermostable DNA polymerase comprises a DNA polymerasefrom the thermophilic bacterium designated Tsp JS1. See, e.g., US2004/0005573 A1. Certain fragments and variants of a thermostable DNApolymerase from Tsp JS1 are known to those skilled in the art. Id.

C. Certain Reverse Transcriptases

Reverse transcriptases are polymerases that can use RNA as a template.Thus, reverse transcriptases catalyze the synthesis of DNA using RNA asa template. In certain instances, reverse transcriptases catalyze DNAusing DNA as the template. As discussed above, certain DNA polymeraseshave reverse transcriptase activity as well.

In certain embodiments, a reverse transcriptase is used to synthesizecDNA from messenger RNA. Thus, in certain embodiments, reversetranscriptases are used in methods that measure gene expression. Certainsuch methods include, but are not limited to, reverse transcriptase PCR(RT-PCR) and microarray analysis. In certain embodiments, reversetranscriptases are used to generate cDNA for sequencing, gene cloning,protein expression, and/or cDNA library construction. In certainembodiments, reverse transcriptases are used in sequence detection whenthe target(s) are RNA. Certain such targets include, but are notlimited, to RNA viruses. In certain embodiments, reverse transcriptasesare used in in vitro nucleic acid amplification techniques that employan RNA intermediate. Certain such exemplary techniques include, but arenot limited to, Ribo-SPIA (Single Primer Isothermal Amplification;NuGEN, San Carlos, Calif.), NASBA/NucliSense (Nucleic Acid SequenceBased Amplification; bioMerieux USA, Durham, N.C.) and TMA(Transcription Mediated Amplification; GenProbe, San Diego, Calif.)technologies.

Certain exemplary classes of reverse transcriptases include, but are notlimited to, reverse transcriptases from avian myeloblastosis virus(AMV), reverse transcriptases from the Moloney murine leukemia virus(MMLV) RT, and Family A DNA polymerases from various bacteria. ExemplaryFamily A DNA polymerases include, but are not limited to, Tth polymerasefrom Thermus thermophilus; Taq polymerase from Thermus aquaticus;Thermus thermophilus Rt41A; Dictyoglomus thermophilum RT46B.1;Caldicellulosiruptor saccharolyticus Tok7B.1; Caldicellulosiruptor spp.Tok13B.1; Caldicellulosiruptor spp. Rt69B.1; Clostridiumthermosulfurogenes; Thermotoga neapolitana; Bacillus caldolyticus EA1.3;Clostridium stercorarium; and Caldibacillus cellulovorans CA2. Shandilyaet al., Extremophiles, 8:243-251 (2004) discusses certain bacterial DNApolymerases with reverse transcriptase activity.

Reverse transcriptases from AMV and MMLV include RNase H domains, whichmediate the degradation of the RNA component of RNA:DNA complexes. Incertain instances, that RNase H activity can decrease the amount offinal product because of the degradation of RNA template. Point mutantsin the RNase H domain of MMLV reverse transcriptase (for example,Superscript II and III, Invitrogen; Powerscript, Takara) and a deletionmutant of the MMLV reverse transcriptase RNase H domain (Superscript I,Invitrogen) are available. In certain instances, deletion of the RNase Hdomain results in severe processivity defects and impaired interactionof the reverse transcriptase with primer-template (see, for example,Telesnitsky et al., Proc. Natl. Acad. Sci. USA, 90:1276-1280 (1993).

In certain instances, an obstacle to generating consistent, full lengthcDNAs in short time periods arises from the inherent propensity of RNAto form secondary structure. In certain instances, regions of secondarystructure in the template RNA can cause reverse transcriptases to stall,fall off the template, or skip over looped out regions. In certaininstances, this can be partially alleviated by running the reversetranscriptase reaction at higher temperatures at which secondarystructures melt. AMV reverse transcriptases and Tth DNA polymerases havebeen used for such higher temperature reactions in view of theirthermostability. In certain instances, nucleic acid binding polypeptideis added in trans to increase polymerase processivity through regions ofRNA secondary structure (see, for example PCT Application WO 0055307).

D. Certain Fusion Proteins

In certain embodiments, fusion proteins are provided. In certain suchembodiments, a fusion protein comprises a nucleic acid bindingpolypeptide and a nucleic acid modification enzyme. In certain suchembodiments, the nucleic acid modification enzyme comprises a nucleicacid polymerase. In certain embodiments, the nucleic acid polymerasecomprises a DNA polymerase. In certain such embodiments, the nucleicacid modification enzyme comprises a reverse transcriptase. In variousembodiments, fusion proteins may comprise any of the nucleic acidbinding polypeptides and any of the polymerases or reversetranscriptases discussed herein.

In certain embodiments, fusion proteins comprising a polymerase and anucleic acid binding polypeptide are provided. In certain suchembodiments, fusion proteins have polymerase activity, exhibitingimproved performance and/or increased efficiency in nucleic acidamplification reactions compared to polymerase alone. In certainembodiments, methods are provided for using fusion proteins in nucleicacid amplification reactions, such as PCR. In certain such embodiments,fusion proteins demonstrate unexpected properties under fast cyclingconditions, having the ability to produce substantial yields ofamplification product. In certain embodiments, fusion proteinscomprising a polymerase and a nucleic acid binding polypeptide can beused in amplification reactions at high pH, for example, at a pH isequal to or greater than 8.5. In certain embodiments, fusion proteinscomprising a polymerase and a nucleic acid binding polypeptide can beused in amplification reactions at high pH, for example, at a pH in therange of 8.5 to 10 (including all pH values between those endpoints). Incertain embodiments, fusion proteins comprising a polymerase and anucleic acid binding polypeptide can be used in amplification reactionsat high pH, for example, at a pH in the range of 8.5 to 9.5.

In certain embodiments, fusion proteins comprising a nucleic acidbinding protein and a given DNA polymerase can be used for RNA-templatedDNA synthesis when the given DNA polymerase alone cannot perform DNApolymerization using a primed RNA template. In certain such embodiments,the DNA polymerase in the fusion protein is a Family B polymerase.

In certain embodiments, fusion proteins comprising a nucleic acidbinding protein and a given DNA polymerase that has reversetranscriptase activity have improved properties compared to the givenDNA polymerase alone. In certain embodiments, fusion proteins comprisinga nucleic acid binding protein and a given reverse transcriptase haveimproved properties compared to the given reverse transcriptase alone.In certain embodiments, the improved properties include one or more ofthe following: improved processivity; the ability to produce longeramplification products; increased ability to read through RNA secondarystructure; shorter reaction times; increased sensitivity; increasedaffinity for a primed template; faster product accumulation; andincreased salt tolerance.

In various embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a nucleic acid modification enzyme, such aspolymerase or reverse transcriptase, is produced using recombinantmethods. In certain such embodiments, a polynucleotide encoding anucleic acid binding polypeptide and a polynucleotide encoding a nucleicacid modification enzyme, such as polymerase or reverse transcriptase,are ligated together in the same reading frame, resulting in apolynucleotide encoding a fusion protein.

In certain embodiments, a polynucleotide encoding a nucleic acid bindingpolypeptide is obtained as described in Part V.A above.

In certain embodiments, a polynucleotide encoding a polymerase or areverse transcriptase is obtained by the polymerase chain reaction(PCR). Certain methods employing PCR are known to those skilled in theart. In certain embodiments, a polynucleotide comprising all or aportion of the coding sequence of a polymerase or a reversetranscriptase is amplified using appropriate primers. In certainembodiments, restriction enzyme sites are included in the primers tofacilitate cloning of the amplification product into an appropriatevector. Certain polynucleotide sequences encoding certain DNApolymerases are known to those skilled in the art. See, e.g., Ito et al.(1991) Nuc. Acids. Research 19:4045-4057; Braithwaite et al. (1993) Nuc.Acids. Research 21:787-802; and Fileé et al. (2002) J. Mol. Evol.54:763-773.

In certain embodiments, a polynucleotide encoding a DNA polymerase is apolynucleotide encoding Taq DNA polymerase (SEQ ID NO:31) or a fragmentor variant thereof having polymerase activity. In certain embodiments, apolynucleotide encoding a DNA polymerase is a polynucleotide encodingPfu DNA polymerase (SEQ ID NO:30) or a fragment or variant thereofhaving polymerase activity. In certain embodiments, a polynucleotideencoding a reverse transcriptase is a polynucleotide encoding the MMLVreverse transcriptase shown in SEQ ID NO:52 or a fragment or variantthereof having polymerase activity.

In various embodiments, a polynucleotide encoding a fusion protein iscloned into a suitable vector. In certain embodiments, a polynucleotideencoding a nucleic acid binding polypeptide and a polynucleotideencoding a nucleic acid modification enzyme, such as polymerase orreverse transcriptase, are ligated together in the same reading frame,and the ligation product is cloned into a suitable vector. In certainembodiments, a polynucleotide encoding a nucleic acid bindingpolypeptide and a polynucleotide encoding a nucleic acid modificationenzyme, such as polymerase or reverse transcriptase, are cloned stepwiseinto a suitable vector.

In certain embodiments, a vector comprising a polynucleotide encoding afusion protein is transferred (e.g., transformed or transfected) into asuitable host cell. Certain exemplary host cells include, but are notlimited to, prokaryotes, yeast cells, insect cells, plant cells, andmammalian cells. See, e.g., Ausubel et al. (1991) Current Protocols inMolecular Biology, Chapter 16, John Wiley & Sons, New York. In certainembodiments, the fusion protein is expressed in the host cell. Incertain such embodiments, the fusion protein is isolated from the hostcell.

In certain embodiments, a suitable vector is an expression vector.Certain expression vectors for the inducible expression of recombinantproteins are known to those skilled in the art. For example, in certainembodiments, a polynucleotide encoding a fusion protein is cloned intoan expression vector such that its transcription is under the control ofan inducible promoter, such as the T7 bacteriophage promoter, the T5promoter, or the tac promoter. See, e.g., the pET series of vectors(Invitrogen, Carlsbad, Calif.), the pQE series of vectors (Qiagen,Valencia, Calif.), or the pGEX series of vectors (Amersham Biosciences,Piscataway, N.J.). Certain such expression vectors are suitable for theexpression of a recombinant protein in a prokaryotic organism.

In certain embodiments, a recombinant expression vector is transformedinto bacteria, such as E. coli. In certain embodiments, expression ofthe fusion protein is induced by culturing the bacteria under certaingrowth conditions. For example, in certain embodiments, expression ofthe fusion protein is induced by addition of isopropylthio-β-galactoside(IPTG) to the culture medium.

In various embodiments of expression vectors, a polynucleotide encodinga tag, such as an affinity tag, is expressed in frame with apolynucleotide encoding a fusion protein. In certain embodiments,certain such tags can provide a mechanism for detection or purificationof the fusion protein. Examples of tags include, but are not limited to,polyhistidine tags, which allow purification using nickel chelatingresin, and glutathione S-transferase moieties, which allow purificationusing glutathione-based chromatography. In certain embodiments, a tag isdisposed at the N-terminus or C-terminus of a fusion protein. In certainembodiments, a tag is disposed internally within a fusion protein.

In certain embodiments, an expression vector further provides a cleavagesite between the tag and the fusion protein, so that the fusion proteinmay be cleaved from the tag following purification. In certainembodiments, e.g., embodiments using polyhistidine tags, the fusionprotein is not cleaved from the tag. It has been reported that thepresence of a polyhistidine tag on a recombinant DNA binding protein mayenhance the interaction of the DNA binding protein with DNA. See, e.g.,Buning et al. (1996) Anal. Biochem. 234:227-230. In certain embodiments,a tag comprises from 1 to 15 histidine residues, including all pointsbetween those endpoints. In certain such embodiments, an increasingnumber of histidine residues is unexpectedly correlated with improvedperformance of the fusion protein in nucleic acid amplificationreactions.

In certain embodiments of a fusion protein, a nucleic acid bindingpolypeptide is joined to the N-terminus of a nucleic acid modificationenzyme. In certain embodiments of a fusion protein, a nucleic acidbinding polypeptide is joined to the C-terminus of a nucleic acidmodification enzyme. In certain embodiments of a fusion protein, anucleic acid binding polypeptide is disposed internally within a nucleicacid modification enzyme.

In certain embodiments of a fusion protein, a nucleic acid bindingpolypeptide is joined to the N-terminus of a reverse transcriptase. Incertain embodiments of a fusion protein, a nucleic acid bindingpolypeptide is joined to the C-terminus of a reverse transcriptase. Incertain embodiments of a fusion protein, a nucleic acid bindingpolypeptide is disposed internally within a reverse transcriptase.

In certain embodiments of a fusion protein, a nucleic acid bindingpolypeptide is joined to the N-terminus of a polymerase. In certainembodiments of a fusion protein, a nucleic acid binding polypeptide isjoined to the C-terminus of a polymerase. In certain embodiments of afusion protein, a nucleic acid binding polypeptide is disposedinternally within a polymerase. Certain three dimensional structures ofcertain DNA polymerases are known to those skilled in the art. See,e.g., Steitz (1999) J. Biol. Chem. 274:17395-17398; Alba (2001) GenomeBiol. 2:3002.1-3002.4. Certain DNA polymerases typically have a“hand-like” three-dimensional structure comprising “finger,” “palm,” and“thumb” domains. See, e.g., Steitz (1999) J. Biol. Chem.274:17395-17398; Alba (2001) Genome Biol. 2:3002.1-3002.4. In certainembodiments of a fusion protein, wherein a nucleic acid bindingpolypeptide is disposed internally within a DNA polymerase, the nucleicacid binding polypeptide occurs within a loop in the “thumb” domain ofthe DNA polymerase. See, e.g., U.S. Pat. No. 5,972,603, e.g., FIG. 4.

In certain embodiments, one skilled in the art can routinely determinewhether a DNA polymerase retains polymerase activity in the context of afusion protein by assaying the fusion protein for polymerase activity.

In certain embodiments, a nucleic acid binding polypeptide is joined toa a nucleic acid modification enzyme, such as polymerase or reversetranscriptase, by chemical methods. In certain embodiments, a nucleicacid binding polypeptide is joined to a nucleic acid modificationenzyme, such as polymerase or reverse transcriptase, by a chemicalcoupling agent. Certain such methods are known to those skilled in theart. See, e.g., Hermanson, ed., Bioconjugate Techniques (Academic Press1996).

In certain embodiments, a nucleic acid binding polypeptide is joined toa a nucleic acid modification enzyme, such as polymerase or reversetranscriptase, by a linker. In certain embodiments, a linker is apeptide, which is joined by peptide bonds to a nucleic acid bindingpolypeptide and to a nucleic acid modification enzyme, such aspolymerase or reverse transcriptase. In certain embodiments, a linker isengineered into a fusion protein by standard recombinant methods. Forexample, in certain embodiments, a polynucleotide encoding a fusionprotein is constructed, wherein a polynucleotide encoding a linker is inframe with and disposed between a polynucleotide encoding a nucleic acidbinding polypeptide and a polynucleotide encoding a nucleic acidmodification enzyme, such as polymerase or reverse transcriptase.

In certain embodiments, a linker is any whole number of amino acids lessthan or equal to 25. In certain embodiments, a linker does not form anα-helix or β-strand. In certain such embodiments, a linker forms anextended, or “loop,” conformation. In certain embodiments, a linkersequence comprises one or more glycine residues. In certain embodiments,a suitable linker sequence is determined using the LINKER program. See,e.g., Crasto et al. (2000) Protein Eng. 13:309-312.

Other exemplary linkers include, but are not limited to, carbohydratelinkers, lipid linkers, fatty acid linkers, and polymeric linkers.Certain exemplary polymeric linkers include, but are not limited to,polyether linkers, such as polyethylene glycol (PEG).

In certain embodiments, full length MMLV reverse transciptase, afragment of MMLV reverse transcriptase, or other mutant forms of reversetranscriptase are cloned into an expression vector. An nonlimitingexemplary expression vector is pET16b (Novagen/EMD Biosciences, LaJolla, Calif.). Exemplary fragments of MMLV reverse transcriptaseinclude, but are not limited to, forms that contain amino acids 1-516(an RNase H deletion form), forms that contain amino acids 1-498 (anRNase H deletion form), and forms that contain amino acids 1 to 360 (anRNase H deletion and connectin domain deletion form). Exemplary mutantsof MMLV reverse transcriptase include, but are not limited to, a form inwhich glutamic acid at position 524 is changed to asparagines (D524N) (aform that decreases RNase H activity) (see, for example, Blain et al.,J. Biol. Chem., 31:23585-23592 (1993)). FIG. 6 shows the MMLV RTpolymerase domain (Pol), the connection domain (Conn), and the RNase Hdomain (RNaseH) of MMLV reverse transcriptase. Amino acids 2 to 672correspond to amino acids 122 to 792 of the MMLV pol polyproteinsequence.

In certain embodiments, the full length, fragment, or mutant form ofMMLV reverse transcriptase in an expression vector is cloned in framewith a nucleic acid binding polypeptide, such as Pae3192, for expressionof a fusion protein. In certain embodiments, the nucleic acid bindingpolypeptide is placed at the N-terminus of the full length, fragment, ormutant form of MMLV reverse transcriptase. In certain embodiments, thenucleic acid binding polypeptide is placed at the C-terminus of the fulllength, fragment, or mutant form of MMLV reverse transcriptase. Incertain embodiments, the expression vector encoding the fusion proteinincludes a tag for affinity purification.

In various embodiments, fusion proteins that comprise a nucleic acidbinding polypeptide and the full length, fragment, or mutant form ofMMLV reverse transcriptase can be subjected to various in vitro assays.Exemplary assays include, but are not limited to, tests for reversetranscriptase activity, including, but not limited to, radioactivenucleotide incorporation and gel analysis of product length and yield.In certain such embodiments, temperature and salt tolerance can also bedetermined. In certain embodiments, the ability of the fusion protein toread through RNAs with significant secondary structure, such as stemloops containing CUUCGG hairpins, is tested. In certain suchembodiments, temperature and salt tolerance is also tested. In certainembodiments, processivity of the fusion protein is assayed usingfluorescently-labeled primers and capillary electrophoresis.

E. Certain Methods Using Nucleic Acid Binding Polypeptides

Example K below shows that Pae3192 not only binds to DNA:DNA duplexes,but also binds to DNA:RNA duplexes. Thus, Ape3192,Sso7d, and othernucleic acid binding polypeptides should also bind to both DNA:DNAduplexes and DNA:RNA duplexes. Accordingly, all of the methods discussedin this Part (Part V.E) in various embodiments may involve a DNA:DNAduplex, a DNA:RNA duplex, or both a DNA:DNA duplex and a DNA:RNA duplex.

1. Stabilize Nucleic Acid Duplexes

In certain embodiments, one or more nucleic acid binding polypeptidesare used to stabilize a nucleic acid duplex from denaturation attemperatures above the Tm of the nucleic acid duplex, therebyeffectively increasing the Tm of the nucleic acid duplex. In certainsuch embodiments, one or more nucleic acid binding polypeptides arecombined with a nucleic acid duplex. In certain such embodiments, theratio of the concentration of a nucleic acid binding polypeptide to theconcentration of the nucleic acid duplex (in nucleotides) is at leastabout 1:25, 1:10, 1:5, 1:3, 1:1, or any ratio wherein the concentrationof the nucleic acid binding polypeptide exceeds that of the nucleic acidduplex.

2. Anneal Complementary Nucleic Acid Strands

In certain embodiments, one or more nucleic acid binding polypeptidesare used to promote the annealing of complementary nucleic acid strands.In certain embodiments, annealing takes place with greater rapidity andspecificity in the presence of a nucleic acid binding polypeptide thanin the absence of a nucleic acid binding polypeptide. In certainembodiments, complementary nucleic acid strands are allowed to anneal ina composition comprising one or more nucleic acid binding polypeptides.In certain such embodiments, a nucleic acid binding polypeptide ispresent at any concentration from about 1 μg/ml to about 500 μg/ml. Incertain embodiments, one or more nucleic acid binding polypeptides areused to favor the annealing of nucleic acid strands that arecomplementary without mismatches over the annealing of nucleic acidstrands that are complementary with mismatches.

In certain embodiments, nucleic acid binding polypeptides are used inhybridization-based detection assays or primer extension assays in whicha probe or primer is annealed to a target nucleic acid sequence. Certainexamples of the use of nucleic acid binding polypeptides in certain suchassays are provided below.

a) Hybridization-Based Detection Assays

In certain embodiments, one or more nucleic acid binding polypeptidesare used to increase the efficiency, e.g., the speed and specificity, ofa hybridization-based detection assay. Exemplary hybridization-baseddetection assays include, but are not limited to, assays in which targetnucleic acid is immobilized on a solid support and exposed to a labeledprobe (see, e.g., Sambrook et al. (2001) Molecular Cloning: A LaboratoryManual (3^(rd) ed., Cold Spring Harbor Laboratory Press, NY), e.g., at6.33-6.58 (describing “Southern” hybridizations). In certainembodiments, exemplary hybridization-based detection assays includemicroarray-based assays in which target nucleic acid is labeled andexposed to a plurality of polynucleotides immobilized on a solidsupport. See id. Appendix 10. An example of the use of the nucleic acidbinding polypeptide Sso7d in a microarray-based detection assay isdescribed, e.g., in Hatakeyama, US 2003/0022162 A1.

In certain hybridization-based detection assays, a nucleic acid probe isexposed to a mixture of nucleic acids. Within that mixture is a targetnucleic acid, which comprises a sequence that is complementary to theprobe. The probe specifically anneals to the target nucleic acid to forma hybridization complex under certain conditions, e.g., conditions inwhich the probe is exposed to the target nucleic acid for an appropriatelength of time and at an annealing temperature below that of thepredicted Tm of the probe.

In certain embodiments, one or more nucleic acid binding polypeptidesare used to increase the Tm of a probe, thereby increasing thetemperature at which the annealing may be carried out. In certain suchembodiments, the annealing is carried out in the presence of one or morenucleic acid binding polypeptides. In certain such embodiments, theannealing takes place at any temperature from 10° C. below to 40° C.above the predicted Tm of the probe. In certain such embodiments, theannealing takes place at a temperature up to 40° C. above the predictedTm of the probe. In certain embodiments in which a probe is anoligonucleotide of about 15-35 nucleotides, annealing takes place in thepresence of one or more nucleic acid binding polypeptides at anytemperature between 40° C. and 85° C.

In certain embodiments, one or more nucleic acid binding polypeptidesare used to increase the Tm of a probe, thereby allowing the use ofshorter probes. In certain such embodiments, the annealing is carriedout in the presence of one or more nucleic acid binding polypeptides. Incertain such embodiments, a probe is of any length between 12 and 25nucleotides. In certain such embodiments, a probe is of any lengthbetween 12 and 19 nucleotides. In certain such embodiments, a probe isof any length between 12 and 16 nucleotides.

In certain embodiments, one or more nucleic acid binding polypeptidesare used to decrease the duration of time to achieve annealing. Incertain such embodiments, the annealing is carried out in the presenceof one or more nucleic acid binding polypeptides. In certain suchembodiments, the annealing takes place over any amount of time fromabout 0.5 minute to about three hours. In certain such embodiments, theannealing takes place over any amount of time from about 1 minute toabout 30 minutes. In certain such embodiments, the annealing takes placeover any amount of time from about 1 minute to about 15 minutes.

In certain embodiments of hybridization-based detection assays, a probemay selectively hybridize to a target nucleic acid that is complementarywithout mismatches to the probe. In certain embodiments, a probe mayalso selectively hybridize to a target nucleic acid that iscomplementary to the probe but that contains one or more mismatchesrelative to the probe. In certain embodiments, one or more nucleic acidbinding polypeptides are used to favor the hybridization of a probe to atarget nucleic acid that is complementary without mismatches to theprobe over the hybridization of a probe to a target nucleic acid that iscomplementary but that contains one or more mismatches relative to theprobe. Thus, in certain embodiments, the specificity of hybridization isincreased. In certain such embodiments, annealing is carried out underany of the conditions of time or temperature described above. In certainsuch embodiments, annealing is carried out at a temperature greater thanthe predicted Tm of the probe.

In certain embodiments, because nucleic acid binding polypeptides cansubstantially increase the speed and specificity of ahybridization-based detection assay, such polypeptides can be used incertain hybridization-based “point-of-use” devices. Point-of-use devicesare typically portable devices that allow rapid diagnosis or detectionof a physiological or pathological condition, in certain instances, in anon-clinical or small-scale laboratory setting. An exemplarypoint-of-use device is, for example, a typical pregnancy test. Anexemplary point-of-use device that uses hybridization-based detectionis, for example, the Affirm VPIII Microbial Identification System(Becton Dickinson and Company—BD Diagnostics, Sparks, Md.), whereby thepresence of certain vaginal pathogens is detected in vaginal swabspecimens using an oligonucleotide hybirdization assay. See Briselden etal. (1994) J. Clin. Microbiol. 32:148-52; Witt et al. (2002) J. Clin.Microbiol. 40:3057-3059.

In certain embodiments, one or more nucleic acid binding polypeptidescan be used in a hybridization-based point-of-use device that diagnosesa pathological condition, such as an infection, by detecting geneticmaterial from a pathogen in a biological sample from a host. In certainembodiments, the volume of a biological sample to be used with apoint-of-use device is reduced in the presence of one or more nucleicacid binding polypeptides. In certain embodiments, thehybridization-based point-of-use device utilizes microarray technology.

In certain embodiments, because nucleic acid binding polypeptides cansubstantially increase the specificity of a hybridization-baseddetection assay, one or more nucleic acid binding polypeptides can beused in assays that detect mutations or polymorphisms in a targetpolynucleotide. For example, one or more nucleic acid bindingpolypeptides can be used in assays that detect single nucleotidepolymorphisms (SNPs). For a review of SNP detection methods, see, e.g.,Shi (2001) Clinical Chem. 47:164-172. In certain embodiments, one ormore nucleic acid binding polypeptides are used in assays that detectrare copies of a target polynucleotide in a complex mixture of nucleicacids. For example, in certain such embodiments, the targetpolynucleotide comprises genetic material from a pathogen containedwithin a biological sample from a host.

b) Increase Tm of Primers in Primer Extension Reactions

In certain embodiments, one or more nucleic acid binding polypeptidesare used to increase the Tm of a primer in a primer extension reaction.In certain primer extension reactions, such as PCR, one or more primersare annealed to a template nucleic acid. In PCR, e.g., the annealingtypically takes place over 30 seconds at about 55° C., a temperaturethat is less than the predicted Tm of a typical primer of about 20-30nucleotides. Sambrook et al. (2001) Molecular Cloning: A LaboratoryManual (3^(rd) ed., Cold Spring Harbor Laboratory Press, NY) at 8.22.

In certain embodiments, one or more nucleic acid binding polypeptidesare used to increase the Tm of a primer in a primer extension reaction,thereby increasing the temperature at which the annealing may be carriedout. In certain such embodiments, the annealing is carried out in thepresence of one or more nucleic acid binding polypeptides. In certainsuch embodiments, the annealing is carried out at any temperature fromabout 55° C. up to about 75° C. In certain such embodiments, theannealing is carried out at any temperature between 60° C. and 70° C. Incertain embodiments, increased annealing temperature reduces certainprimer artifacts, such as primer dimers and hairpin formation.

In certain embodiments, one or more nucleic acid binding polypeptidesare used to increase the Tm of a primer in a primer extension reaction,thereby allowing the use of shorter primers. In certain suchembodiments, the annealing is carried out in the presence of one or morenucleic acid binding polypeptides. In certain such embodiments, a primeris of any length between 12 and 19 nucleotides. In certain suchembodiments, a primer is of any length between 12 and 16 nucleotides.

3. Enhance Activity of Nucleic Acid Modification Enzymes

In certain embodiments, one or more nucleic acid binding polypeptidesare used to enhance the activity of a nucleic acid modification enzyme.In certain such embodiments, one or more nucleic acid bindingpolypeptides are included in a composition comprising a nucleic acidmodification enzyme and a nucleic acid, thus enhancing the activity ofthe nucleic acid modification enzyme. In various embodiments, theenhancement in the activity of a nucleic acid modification enzyme isdemonstrated by comparing the activity of the nucleic acid modificationenzyme in the presence of one or more nucleic acid binding polypeptideswith its activity in the absence of one or more nucleic acid bindingpolypeptides. In certain embodiments, the following assays may be usedto evaluate the activity of a nucleic acid modification enzyme:

In certain embodiments, the activity of a gyrase or topoisomerase isassessed by determining the change in the supercoiled state of a nucleicacid exposed to the gyrase or topoisomerase in the presence and in theabsence of one or more nucleic acid binding polypeptides.

In certain embodiments, the activity of a nuclease is assessed bydetermining the amount of cleavage product produced by the nuclease inthe presence and in the absence of one or more nucleic acid bindingpolypeptides. In certain such embodiments, the activity of a restrictionendonuclease is assessed by exposing a nucleic acid to a restrictionendonuclease in the presence and in the absence of one or more nucleicacid binding polypeptides. In certain such embodiments, the extent ofdigestion by the restriction endonuclease is determined by gelelectrophoresis.

In certain embodiments, the activity of a methylase is determined byassessing the methylation state of a nucleic acid exposed to a methylasein the presence and in the absence of one or more nucleic acid bindingpolypeptides. In certain such embodiments, the methylation state of thenucleic acid is assessed, for example, by determining the extent towhich the nucleic acid is cleaved by a methylation sensitive restrictionendonuclease, such as MboI.

In certain embodiments, the activity of a ligase is assessed bydetermining the amount of ligation product produced by the ligase in thepresence and in the absence of one or more nucleic acid bindingpolypeptides. In certain such embodiments, a circularized plasmid islinearized by a restriction endonuclease, isolated from the restrictionendonuclease, and exposed to ligase in the presence and in the absenceof one or more nucleic acid binding polypeptides. In certain suchembodiments, the ligation reaction mixture is used to transformcompetent bacteria. In certain such embodiments, the number oftransformants is proportional to the activity of the ligase.

In certain embodiments, the activity of a polymerase is assessed in thepresence and in the absence of one or more nucleic acid bindingpolypeptides using a polymerase activity assay described above.

4. Increase Processivity of a DNA Polymerase

In certain embodiments, one or more nucleic acid binding polypeptidesare used to improve the performance of DNA polymerase. In certain suchembodiments, improved performance of DNA polymerase is increasedprocessivity of the DNA polymerase in a primer extension reaction. Incertain embodiments, the primer extension reaction is PCR. For example,in certain embodiments, the inclusion of one or more nucleic acidbinding polypeptides in a PCR reaction allows for more efficientamplification of targets under suboptimal conditions, such as high saltconcentrations. Examples of certain high salt concentrations includefrom 60 mM KCl to 130 mM KCl for Taq DNA polymerase, and from 40 mM KClto 130 mM KCl for Pfu polymerase. In certain embodiments, the inclusionof one or more nucleic acid binding polypeptides in a PCR reactiondecreases the time of the extension step of PCR to, for example, ≦5minutes, ≦3 minutes, ≦2 minutes, ≦1 minute, or ≦30 seconds. In certainembodiments, the inclusion of one or more nucleic acid bindingpolypeptides in a PCR reaction allows for more efficient amplificationof long targets, for example, targets from about 5 kb to about 20 kb.

F. Certain Methods Using Fusion Proteins

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a nucleic acid modification enzyme is used inany method that uses a nucleic acid binding polypeptide (as described,for example, in Part V.E. above), except that the fusion proteinreplaces the nucleic acid binding polypeptide in the method. In certainembodiments, a fusion protein comprising a nucleic acid bindingpolypeptide and a nucleic acid modification enzyme is used in any methodthat uses a nucleic acid binding polypeptide (as described, for example,in Part V.E. above), except that the fusion protein is used incombination with the nucleic acid binding polypeptide in the method.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a nucleic acid modification enzyme is used inany reaction in which the nucleic acid modification enzyme alone can beused. In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a nucleic acid modification enzyme is used toimprove the efficiency of any reaction in which the nucleic acidmodification enzyme alone can be used. In certain such embodiments, afusion protein comprising a nucleic acid binding polypeptide and anucleic acid modification enzyme has increased activity relative to thenucleic acid modification enzyme alone. In certain such embodiments, theassays set forth in Part V.E.3 above may be used to evaluate theactivity of a nucleic acid modification enzyme or a fusion proteincomprising a nucleic acid binding polypeptide and a nucleic acidmodification enzyme. In certain embodiments, a fusion protein comprisinga nucleic acid binding polypeptide and a DNA polymerase has increasedprocessivity relative to the DNA polymerase alone.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a DNA polymerase is used in a primer extensionreaction. In certain such embodiments, the fusion protein increases theefficiency of the primer extension reaction. In certain embodiments, afusion protein comprising a nucleic acid binding polypeptide and a DNApolymerase is included in a primer extension reaction to increase the Tmof one or more primers in the reaction. In certain embodiments, thetemperature at which annealing is carried out may be increased. Incertain embodiments, shorter primers may be used.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a thermostable DNA polymerase is included in aPCR reaction. In certain such embodiments, the fusion protein increasesthe efficiency of PCR. In certain embodiments, a fusion proteincomprising a nucleic acid binding polypeptide and a thermostable DNApolymerase is included in a PCR reaction that is conducted undersuboptimal conditions, such as high salt concentrations. Exemplary highsalt concentrations are described above in Part V.E.4. In certainembodiments, a fusion protein comprising a nucleic acid bindingpolypeptide and a thermostable DNA polymerase is included in a PCRreaction to decrease the time of the extension step of PCR. Exemplaryextension times are provided above in Part V.E.4. In certainembodiments, a fusion protein comprising a nucleic acid bindingpolypeptide and a thermostable DNA polymerase is included in a PCRreaction to more efficiently amplify long targets. Exemplary targetlengths are provided above in Part V.E.4. In certain embodiments, afusion protein comprising a nucleic acid binding polypeptide and athermostable DNA polymerase is included in a PCR reaction to increasethe amount of PCR amplification product.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a thermostable DNA polymerase is used in “hotstart” PCR. In certain embodiments, “hot start” PCR is used to suppressnon-specific binding of primer to template. See, e.g., Sambrook et al.(2001) Molecular Cloning: A Laboratory Manual (3^(rd) ed., Cold SpringHarbor Laboratory Press, NY) at 8.110 (describing “hot start” PCR). Incertain embodiments of “hot start” PCR, one or more components to beused in a PCR are prevented from functioning in the PCR until thereaction mixture reaches or exceeds a temperature at which non-specificpriming does not occur. Id. For example, in certain embodiments of “hotstart” PCR, an antibody to the thermostable DNA polymerase is used toreversibly block polymerase activity until a suitable temperature isreached. See, e.g., Kellogg et al. (1994) Biotechniques 16:1134-1137(describing the use of antibodies to Taq DNA polymerase). In certainembodiments, a fusion protein comprising a nucleic acid bindingpolypeptide and a thermostable DNA polymerase is used in “hot start”PCR. In certain such embodiments, an antibody to the nucleic acidbinding polypeptide is used to reversibly block nucleic acid bindingactivity and/or polymerase activity until a suitable temperature isreached.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a reverse transcriptase is used in a primerextension reaction. In certain such embodiments, the fusion proteinincreases the efficiency of the primer extension reaction. In certainembodiments, a fusion protein comprising a nucleic acid bindingpolypeptide and a reverse transcriptase is included in a primerextension reaction to increase the Tm of one or more primers in thereaction. In certain embodiments, the temperature at which annealing iscarried out may be increased. In certain embodiments, shorter primersmay be used.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a thermostable reverse transcriptase is includedin an RT-PCR (reverse transcriptase-PCR) reaction. In certain suchembodiments, the fusion protein increases the efficiency of RT-PCR. Incertain embodiments, a fusion protein comprising a nucleic acid bindingpolypeptide and a thermostable reverse transcriptase is included in aRT-PCR reaction that is conducted under suboptimal conditions, such ashigh salt concentrations. Exemplary high salt concentrations aredescribed above in Part V.E.4. In certain embodiments, a fusion proteincomprising a nucleic acid binding polypeptide and a thermostable reversetranscriptase is included in a RT-PCR reaction to decrease the time ofthe extension step of RT-PCR. Exemplary extension times are providedabove in Part V.E.4. In certain embodiments, a fusion protein comprisinga nucleic acid binding polypeptide and a thermostable reversetranscriptase is included in a RT-PCR reaction to more efficientlyamplify long targets. Exemplary target lengths are provided above inPart V.E.4. In certain embodiments, a fusion protein comprising anucleic acid binding polypeptide and a thermostable reversetranscriptase is included in a RT-PCR reaction to increase the amount ofRT-PCR amplification product.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a thermostable reverse transcriptase is used in“hot start” RT-PCR. In certain embodiments, “hot start” RT-PCR is usedto suppress non-specific binding of primer to template. See, e.g.,Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3^(rd)ed., Cold Spring Harbor Laboratory Press, NY) at 8.110 (describing “hotstart” RT-PCR). In certain embodiments of “hot start” RT-PCR, one ormore components to be used in a RT-PCR are prevented from functioning inthe RT-PCR until the reaction mixture reaches or exceeds a temperatureat which non-specific priming does not occur. Id. For example, incertain embodiments of “hot start” RT-PCR, an antibody to thethermostable reverse transcriptase is used to reversibly block reversetranscriptase activity until a suitable temperature is reached. See,e.g., Kellogg et al. (1994) Biotechniques 16:1134-1137 (describing theuse of antibodies to Taq DNA polymerase). In certain embodiments, afusion protein comprising a nucleic acid binding polypeptide and athermostable reverse transcriptase is used in “hot start” RT-PCR. Incertain such embodiments, an antibody to the nucleic acid bindingpolypeptide is used to reversibly block nucleic acid binding activityand/or reverse transcriptase activity until a suitable temperature isreached.

G. Certain Exemplary Amplification Methods Using Fusion Proteins

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a polymerase is used to amplify a target nucleicacid sequence, e.g., in a primer extension reaction. In certain suchembodiments, the primer extension reaction is PCR. Certain exemplarymethods for performing PCR are known to those skilled in the art. See,e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual(3^(rd) ed., Cold Spring Harbor Laboratory Press, NY) at 8.18-8.24;Innis et al. (1990) PCR Protocols: A Guide to Methods and Applications(Academic Press, NY).

1. “Fast” PCR

In various instances, a typical PCR cycle comprises denaturing adouble-stranded nucleic acid, annealing at least two primers to oppositestrands of the denatured nucleic acid, and extending the primers using athermostable DNA polymerase. In various embodiments, the primers aretypically oligodeoxyribonucleotides of about 18-25 nucleotides inlength. In various instances, the denaturing step is typically at least30 seconds in length at a temperature of at least about 90° C. Invarious instances, the annealing step is typically at least 30 secondsin length at a temperature that is less than the predicted Tm of theprimers. In various instances, the annealing is typically conducted atabout 55° C. for a primer of about 18-25 nucleotides. In variousinstances, the extension step typically takes place at 72° C. for oneminute per 1000 base pairs of target DNA. In various instances, about25-30 cycles are typically performed to generate detectableamplification product. For certain typical PCR conditions, see, e.g.,Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3^(rd)ed., Cold Spring Harbor Laboratory Press, NY) at 8.22.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a polymerase unexpectedly allows for theamplification of a target nucleic acid using substantially fastercycling conditions, e.g., substantially decreased denaturing, annealing,and/or extension times, as described below.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a polymerase stabilizes the primer-templateduplex, thereby increasing the Tm of the primers above the predicted Tm.Accordingly, in certain embodiments, the annealing is carried out at atemperature that is greater than the predicted Tm of the primers. Incertain such embodiments, it is possible to carry out the annealing andextension at the same temperature in a single step, thus increasing theefficiency of PCR.

In certain embodiments, the annealing is carried out at a temperaturethat is from about 1° C. to about 40° C. above the predicted Tm of atleast one of the primers (including all points between those endpoints).In certain such embodiments, the annealing is carried out at about 5°C., 10° C., 15° C., or 20° C. above the predicted Tm of at least one ofthe primers.

In certain embodiments, the annealing is carried out at any temperaturefrom about 55° C. up to about 80° C. (including all points between thoseendpoints). In certain such embodiments, the annealing is carried out atany temperature from about 62° C. to about 78° C.; from about 62° C. toabout 75° C.; from about 65° C. to about 72° C.; from about 65° C. toabout 75° C.; from about 68° C. to about 72° C.; and from about 68° C.to about 75° C. In certain embodiments, the annealing and extension arecarried out at the same temperature.

In certain embodiments, annealing at temperatures higher than theannealing temperatures typically used in PCR may, under certaincircumstances, have other beneficial effects. For example, in certainembodiments, annealing at higher temperatures may improve primerspecificity (i.e., may alleviate “mispriming”). In certain embodiments,annealing at higher temperatures may allow for more efficientamplification of problematic targets, such as targets having repetitivesequences or targets having complex secondary structure, such as GC-richtargets.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a polymerase is used in PCR amplificationshaving substantially decreased denaturing, annealing, and/or extensiontimes. Generally, the time of the denaturing, annealing, and/orextension step in a PCR cycle is measured as the amount of time that thereaction mixture is held at the denaturing, annealing, and/or extensiontemperature once the reaction mixture reaches that temperature. Incertain embodiments, the time of the denaturing, annealing, and/orextension step is any amount of time that is less than or equal to 30seconds. For example, in certain embodiments, the time of thedenaturing, annealing, and/or extension step is less than or equal to30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 second. In certainembodiments, the time of the denaturing, annealing, and/or extensionstep is 0 seconds. In certain embodiments, the annealing and extensionare performed in a single step that is of any of the above lengths oftime.

Exemplary embodiments of a PCR amplification cycle comprising adenaturing step, an annealing step, and an extension step are asfollows. In certain such embodiments, a reaction mixture comprising atarget nucleic acid, at least two primers, and a fusion proteincomprising a polymerase and a nucleic acid binding polypeptide isbrought to a denaturing temperature (a temperature capable of denaturingthe target nucleic acid). Bringing the reaction mixture to thedenaturing temperature encompasses heating or cooling the reactionmixture to the denaturing temperature, or maintaining the reactionmixture at the denaturing temperature without heating or cooling it.After bringing the reaction mixture to the denaturing temperature, thereaction mixture is cooled to an annealing temperature. At the annealingtemperature, the at least two primers are capable of selectivelyhybridizing to opposite strands of the target nucleic acid. In certainembodiments, the annealing temperature is greater than the Tm of atleast one of the primers. After cooling the reaction mixture to theannealing temperature, the reaction mixture is heated to an extensiontemperature. The extension temperature allows for the extension of theat least two primers by the fusion protein.

In certain embodiments of the above PCR amplification cycle, thereaction mixture is held at the denaturing, annealing, and/or extensiontemperature for any amount of time that is less than or equal to 30seconds. For example, in certain embodiments, the reaction mixture isheld at the denaturing, annealing, and/or extension temperature for lessthan or equal to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1second. In certain such embodiments, the reaction mixture is held at thedenaturing, annealing, and/or extension temperature for 0 seconds. Incertain such embodiments, the reaction mixture is cycled from onetemperature to the next without holding at any temperature (i.e., thetime of the denaturing, annealing, and extension steps is 0 seconds).

Exemplary embodiments of a PCR amplification cycle comprising adenaturing step and a combined annealing/extension step are as follows.In certain such embodiments, a reaction mixture comprising a targetnucleic acid, at least two primers, and a fusion protein comprising apolymerase and a nucleic acid binding polypeptide is brought to adenaturing temperature. Bringing the reaction mixture to the denaturingtemperature encompasses heating or cooling the reaction mixture to thedenaturing temperature, or maintaining the reaction mixture at thedenaturing temperature without heating or cooling it. After bringing thereaction mixture to the denaturing temperature, the reaction mixture iscooled to an annealing/extension temperature. In certain embodiments,the annealing/extension temperature is greater than the Tm of at leastone of the primers. At the annealing/extension temperature, the at leasttwo primers selectively hybridize to opposite strands of the denaturedtarget nucleic acid and are extended by the fusion protein.

In certain embodiments of the above PCR amplification cycle, thereaction mixture is held at either the denaturing temperature and/or theannealing/extension temperature for any amount of time that is less thanor equal to 30 seconds. For example, in certain embodiments, thereaction mixture is held at either the denaturing temperature and/or theannealing/extension temperature for less than or equal to 30, 25, 20,15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 second. In certain suchembodiments, the reaction mixture is held at either the denaturingtemperature and/or the annealing/extension temperature for 0 seconds. Incertain such embodiments, the reaction mixture is cycled from thedenaturing temperature to the annealing/extension temperature withoutholding at either temperature (i.e., the time of both the denaturingstep and the combined annealing/extension step is 0 seconds).

In certain embodiments, a target nucleic acid is denatured by exposingthe target nucleic acid to a helicase. See, e.g., Moore (2005) Nature435:235-238. In certain such embodiments, the denaturing step and theannealing step of a PCR amplification cycle may be performed at the sametemperature and/or in a single step. In certain such embodiments, thedenaturing step and the combined annealing/extension step of a PCRamplification cycle are performed at the same temperature and/or in asingle step.

In certain embodiments, a PCR amplification cycle is repeated multipletimes. In various embodiments, the number of cycles may vary. Forexample, in certain embodiments, the number of cycles may relate to theinitial concentration of the target nucleic acid, such that more cyclesare performed for targets initially present at lower concentrations. Incertain embodiments, the number of cycles performed is sufficient togenerate detectable amplification product.

In certain embodiments, the total time to complete a PCR cycle issubstantially decreased. The duration of time to complete a single PCRcycle depends, in part, on the amount of time that the reaction is heldat the denaturing, annealing, and/or extension temperatures. That amountof time may be user-specified, e.g., based on the denaturing, annealing,and extension times that optimize the specificity and/or yield ofamplification product. The duration of time to complete a single PCRcycle also depends, in part, on the amount of time to transition fromone temperature to another (i.e., the “ramping” time). That amount oftime may be user-specified and/or may depend on the instrumentation usedto perform thermal cycling.

The amount of time to complete a single amplification cycle varies amongcertain known thermal cyclers. For example, certain thermal cyclers arecapable of completing a single amplification cycle in about 15 to about45 seconds for reaction volumes of about 10-30 μl. See, e.g., AppliedBiosystems 9800 Fast PCR System, 2004 product overview (AppliedBiosystems, Foster City, Calif.); Roche LightCycler® System (RocheApplied Science, Indianapolis, Ind.); the SmartCycler® System (Cepheid,Sunnyvale, Calif.); the RapidCycler instruments (Idaho Technology, SaltLake City, Utah); and U.S. Pat. No. 6,787,338 B2. Certain thermalcyclers are capable of completing a single amplification cycle in aslittle as 4 to 6 seconds. See, e.g., the PCRJet, Megabase ResearchProducts, Lincoln, Nebr., patented under U.S. Pat. No. 6,472,186; andU.S. Pat. No. 6,180,372 B1. For a review of instrumentation capable ofrapid cycling times, see, e.g., Moore (2005) Nature 435:235-238.

In certain embodiments, the time to complete a single PCR cycle is anyamount of time that is less than or equal to 90 seconds. For example, incertain embodiments, the time to complete a single PCR cycle is lessthan or equal to 90, 75, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5seconds.

In various embodiments, PCR may be carried out in any of a variety ofvessels. Certain such vessels include, but are not limited to, microfugetubes (including thin-walled microfuge tubes); microcapillaries; andmulti-well plates (including thin-walled multi-well plates), such as96-well, 384-well, and 1536-well plates. In certain embodiments, thechoice of vessel depends on the thermal cycler used. Certain exemplarythermal cyclers and suitable vessels for such cyclers are known to thoseskilled in the art, e.g., the GeneAmp® PCR System 9700 and AppliedBiosystems 9800 Fast PCR System (Applied Biosystems, Foster City,Calif.). See also Constans (2001) The Scientist 15(24):32 at pp. 1-7(Dec. 10, 2001); U.S. Pat. Nos. 6,787,338 B2, 6,180,372 B1, 6,640,891B1, 6,482,615 B2, and 6,271,024 B1.

In certain embodiments, amplification products are detected using anynucleic acid detection method. For example, in certain embodiments,amplification products are detected using certain routine gelelectrophoresis methods known to those skilled in the art. In certainembodiments, amplification products are detected using massspectrometry. See, e.g., U.S. Pat. No. 6,180,372. In certainembodiments, amplification products are detected in the reactionmixture, e.g., either during one or more amplification cycles and/orafter completion of one or more amplification cycles. See, e.g., U.S.Pat. Nos. 6,814,934 B1, 6,174,670 B1, and 6,569,627 B2, and Pritham etal. (1998) J. Clin. Ligand Assay 21:404-412. Certain such embodimentsare described below, Part V.G.3. In certain embodiments, amplificationproducts are detected using one or more labeled primers or probes.Certain such primers and probes are described below, Part V.G.3.

2. Certain PCR Conditions

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a polymerase exhibits improved performancerelative to polymerase alone. For example, in certain embodiments, afusion protein comprising a nucleic acid binding polypeptide and apolymerase is capable of amplifying targets in higher saltconcentrations than polymerase alone. Thus, in certain embodiments, saltconcentrations from about 10 mM to about 130 mM (including all pointsbetween those endpoints) may be used. Exemplary salt concentrationsinclude, but are not limited to, about 40, 50, 60, 70, 80, 90, and 100mM of a monovalent salt, such as KCl.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a polymerase is capable of amplifying targets ata higher pH than polymerase alone. Thus, in certain embodiments, the pHmay be equal to or greater than 8.5. In certain embodiments, fusionproteins comprising a polymerase and a nucleic acid binding polypeptidecan be used in amplification reactions at high pH, for example, at a pHin the range of 8.5 to 10 (including all pH values between thoseendpoints). In certain embodiments, fusion proteins comprising apolymerase and a nucleic acid binding polypeptide can be used inamplification reactions at high pH, for example, at a pH in the range of8.5 to 9.5.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a polymerase is capable of amplifying longtargets more efficiently than polymerase alone. Thus, in certainembodiments, a fusion protein comprising a nucleic acid bindingpolypeptide and a polymerase is able to more efficiently amplify targetsfrom at least about 5 kb to at least about 20 kb in length (includingall points between those endpoints).

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a polymerase is capable of producing higheryields of amplification product than polymerase alone under the sameamplification conditions. In certain such embodiments, the yield (amountof amplification product) produced by the fusion protein is from about 2to about 500 fold higher (including all points between those endpoints)than the yield produced by polymerase alone under the same conditions.Accordingly, in certain embodiments, a fusion protein comprising anucleic acid binding polypeptide and a polymerase uses fewer cycles togenerate the same amount of amplification product as polymerase aloneunder the same conditions. In certain embodiments, the number of cyclesin a PCR is from about 15 to about 40 (including all points betweenthose endpoints).

In certain embodiments, yield is calculated by the following equation:N=N₀(1+E)^(n), where N is the number of amplified molecules, N₀ is theinitial number of molecules, n is the number of amplification cycles,and E is the “amplification efficiency.” See Arezi et al. (2003)Analytical Biochem. 321:226-235. “Amplification efficiency” may bedetermined by the following equation: E=10^([−1/slope])−1, where “slope”is the slope of the line of the plot of C_(T) versus the log of theintial target copy number. See id. C_(T) is the “threshold cycle,” orthe cycle in which the emission intensity of the amplification productmeasured by a real-time PCR instrument (such as the 7500 Fast Real-TimePCR System (Applied Biosystems, Foster City, Calif.)) is recorded asstatistically significant above background noise when reactioncomponents are not limiting. See id. In certain instances, amplificationefficiency for a particular polymerase may vary with target length. Seeid.

In certain embodiments, the amplification efficiency of a fusion proteincomprising a nucleic acid binding polypeptide and a polymerase is from0.5 to 1.0 (including all points between those endpoints). In certainembodiments, the amplification efficiency of a fusion protein comprisinga nucleic acid binding polypeptide and a polymerase is from at least 10%to at least 60% greater than that of polymerase alone under the sameconditions.

In certain embodiments, the yield produced by a fusion proteincomprising a nucleic acid binding polypeptide and a polymerase is from85% to 100% (including all points between those endpoints) of thetheoretical maximum possible yield, N=N₀2^(n), which assumes that theamount of product doubles with each amplification cycle. See id. Incertain embodiments, the yield produced by a fusion protein comprising anucleic acid binding polypeptide and a polymerase in a singleamplification cycle is from 1.4N₀ to 2N₀, including all points betweenthose endpoints, where N₀ is the initial number of molecules (i.e., thenumber of molecules present at the start of the amplification cycle). Incertain embodiments, the yield produced by a fusion protein comprising anucleic acid binding polypeptide and a polymerase after n amplificationcycles is from N_(o)(1.4)^(n) to N₀(2)^(n), including all points betweenthose endpoints.

In certain embodiments, as discussed above, a fusion protein comprisinga nucleic acid binding polypeptide and a polymerase increases the Tm ofprimers above the predicted Tm. In certain embodiments, this allows forthe use of primers shorter than those typically used in PCR. Forexample, in certain embodiments, primers may be used that are about 12nucleotides in length or longer. In certain embodiments, exemplaryprimer lengths are from about 12 to about 30 nucleotides (including allpoints between those endpoints).

In certain embodiments, one or more additives that enhance theperformance of a polymerase are added to a PCR. Certain exemplaryadditives are described, e.g., in Sambrook et al. (2001) MolecularCloning: A Laboratory Manual (3^(rd) ed., Cold Spring Harbor LaboratoryPress, NY) at p. 8.9. In certain embodiments, one or more “polymeraseenhancing factors” are added to a PCR to enhance the performance of afusion protein comprising an archaeal family B polymerase (or a fragmentor variant thereof) and a nucleic acid binding polypeptide. Certainexemplary archaeal family B polymerase enhancing factors are described,e.g., in U.S. Pat. No. 6,183,997 B1. In certain embodiments, thepolymerase enhancing factor is a dUTPase.

Exemplary guidance for certain other PCR conditions (e.g., primerconcentration, dNTP concentration, units of polymerase, and targetconcentration) may be found in the art. Certain exemplary conditions areprovided below.

In certain embodiments, the concentration of each PCR primer is fromabout 0.1 μM to about 2.5 μM (including all points between thoseendpoints). In certain embodiments, the concentration of each PCR primeris from about 0.5 to about 1 μM. In certain embodiments, the primers arepresent at different concentrations.

In certain embodiments, at least one primer in a PCR comprises a 3′portion that selectively hybridizes to the target nucleic acid and a 5′portion that does not selectively hybridize to the target nucleic acid.In certain such embodiments, the sequence of the 5′ portion is the sameas the sequence of a “universal” primer. Those skilled in the art arefamiliar with certain universal primers and their use in certainamplification reactions. See, e.g., U.S. Pat. No. 6,270,967 B1; Lin etal. (1996) Proc. Nat'l Acad. Sci. USA 93:2582-2587. In certain suchembodiments, the universal primer may then be used to amplify theamplification products generated by primers that selectively hybridizeto the target nucleic acid.

In certain embodiments, primers are used under conditions that favorasymmetric PCR. According to certain embodiments, an asymmetric PCR mayoccur when (i) at least one primer is in excess relative to the otherprimer(s); (ii) only one primer is used; (iii) at least one primer isextended under given amplification conditions and another primer isdisabled under those conditions; or (iv) both (i) and (iii).Consequently, an excess of one strand of the amplification product(relative to its complement) is generated in asymmetric PCR.

In certain embodiments, primers are used having different Tms. Suchembodiments have been called asynchronous PCR (A-PCR). See, e.g.,published U.S. Patent Application No. US 2003-0207266 A1, filed Jun. 5,2001. In certain embodiments, the Tm of a primer is at least 4-15° C.different from the Tm₅₀ of another primer.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a polymerase has polymerase activity of about0.25 to about 10 units (including all points between those endpoints).In certain such embodiments, polymerase activity is from about 1 toabout 5 units (including all points between those endpoints). In certainsuch embodiments, polymerase activity is from about 1 to about 2.5 units(including all points between those endpoints).

In certain embodiments, the concentration of each dNTP is from about 20to about 500 μM (including all points between those endpoints). Incertain such embodiments, the concentration of each dNTP is about 250μM.

In certain embodiments, the target nucleic acid to be amplified may bein double-stranded form. In certain embodiments, the target nucleic acidto be amplified may be in single-stranded form. In certain embodimentsin which the target nucleic acid is in single-stranded form, the firstamplification cycle can be a linear amplification in which only oneprimer is extended. In certain embodiments, the target nucleic acid maybe present in a sample comprising a complex mixture of nucleic acids andother macromolecules. In certain embodiments, the target nucleic acidmay be present in only a few copies. In certain embodiments, the targetnucleic acid may be present in a single copy.

3. Certain Real-Time PCR

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a polymerase is used to amplify a target nucleicacid using “real-time” PCR. For a review of certain real-time PCR, see,e.g., Edwards et al. (ed.) Real-Time PCR, an Essential Guide (HorizonBioscience, 2004). In certain embodiments of real-time PCR, the progressof the PCR is monitored at any point during or after one or moreamplification cycles and, optionally, after the completion of allamplification cycles. In certain embodiments, the progress of a PCR ismonitored by detecting the presence of amplification products in thereaction. Exemplary methods for performing real-time PCR are described,for example, in U.S. Pat. Nos. 6,814,934 B1, 6,174,670 B1, and 6,569,627B2, and in Pritham et al. (1998) J. Clin. Ligand Assay 21:404-412.Exemplary instruments for performing real-time PCR include, but are notlimited to, the ABR PRISM® 7000 Sequence Detection System; the AppliedBiosystems 7300 Real-Time PCR System, 7500 Real-Time PCR System, 7500Fast Real-Time PCR System, and 7900HT Fast Real-Time PCR System (AppliedBiosystems, Foster City, Calif.); and certain instrumentation discussedabove, Part V.G.1.

In certain embodiments of real-time PCR, the reaction includes anindicator molecule. In certain embodiments, an indicator moleculeindicates the amount of double-stranded DNA in the reaction. In certainsuch embodiments, an indicator molecule is a fluorescent indicator. Incertain such embodiments, a fluorescent indicator is a nucleic acidbinding dye. Certain such dyes include, but are not limited to, SYBR®Green I (see, e.g., U.S. Pat. No. 6,569,627); SYBR® Gold; thiazoleorange; ethidium bromide; pico green; acridine orange; quinolinium4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-diiodide(YOPRO®); quinolinium4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-diiodide(TOPRO®); and chromomycin A3. SYBR® Green I, SYBR® Gold, YOPRO®, andTOPRO® are commercially available from Molecular Probes, Inc., Eugene,Oreg.

In certain embodiments of real-time PCR, a fusion protein comprising anucleic acid binding polypeptide and a polymerase having 5′ to 3′exonuclease activity is used to amplify a target nucleic acid. Incertain embodiments of real-time PCR, a fusion protein comprising anucleic acid binding polypeptide and a polymerase that lacks 5′ to 3′exonuclease is used to amplify a target nucleic acid. In certain suchembodiments, 5′ to 3′ exonuclease activity is provided in trans, e.g.,by including a polypeptide that has 5′ to 3′ exonuclease activity. Incertain embodiments, a polypeptide that has 5′ to 3′ exonucleaseactivity is an enzyme such as a eukaryotic or archaeal “flap”endonuclease, e.g., FEN1. See, e.g., Kaiser et al. (1999) J. Biol. Chem.274:21387-21394. In certain embodiments, a polypeptide that has 5′ to 3′exonuclease activity is a polymerase, such as a bacterial family Apolymerase. In certain such embodiments, the polymerase is a variant ofa bacterial family A polymerase having reduced polymerase activity. Incertain embodiments, a polypeptide that has 5′ to 3′ exonucleaseactivity is a domain isolated from a polymerase, wherein the domain has5′ to 3′ exonuclease activity.

In certain embodiments, real-time PCR is conducted in the presence of anindicator probe. In certain embodiments, an indicator probe produces adetectable signal in the presence of amplification product. In certainembodiments, an indicator probe selectively hybridizes to a strand of anamplification product, resulting in the production of a detectablesignal.

In certain embodiments, an indicator probe is an interaction probecomprising two moieties, wherein one of the moieties is capable ofinfluencing the detectable signal from the other moiety depending uponwhether the probe is hybridized to a strand of an amplification product.For example, in certain such embodiments, one moiety of an interactionprobe is a fluorophore, such that energy from the fluorophore istransferred to the other moiety by the process of fluorescence resonanceenergy transfer (FRET) depending upon whether the probe is hybridized toa strand of the amplification product. In certain embodiments, FREToccurs when the probe is hybridized to a strand of an amplificationproduct. In certain embodiments, FRET occurs when the probe is nothybridized to a strand of an amplification product.

In certain embodiments, an indicator probe is a 5′-nuclease probe. Incertain such embodiments, the probe comprises a fluorophore linked to aquencher moiety through an oligonucleotide link element, wherein energyfrom the fluorophore is transferred to the quencher moiety in the intactprobe through the process of FRET. By this process, fluorescence fromthe fluorophore is quenched. In certain embodiments, the quencher moietyis a different fluorophore that is capable of fluorescing at a differentwavelength. Certain exemplary fluorophores include, but are not limitedto, 6FAM™, VIC™, TET™ or NED™ (Applied Biosystems, Foster City, Calif.).Certain exemplary quencher moieties include, but are not limited to,certain non-fluorescent minor groove binders (MGB) and TAMRA™ (which isalso a fluorophore) (Applied Biosystems, Foster City, Calif.).

In certain embodiments, the 5′-nuclease probe, when hybridized to astrand of the amplification product, is cleaved by the 5′ to 3′exonuclease activity of an extending polymerase and/or by a polypeptidehaving 5′ to 3′ exonuclease activity. In certain embodiments, cleavageis detected by a change in fluorescence. Thus, in certain embodiments,the change in fluorescence is related to the amount of amplificationproduct in the reaction. In certain embodiments in which the 5′-nucleaseprobe comprises a fluorophore linked to a quencher moiety, cleavage ofthe probe results in an increase in fluorescence from the fluorophore.In certain such embodiments in which the quencher moiety is a differentfluorophore, the fluorescence from the quenching moiety is decreased.Certain exemplary methods for using 5′-nuclease probes for the detectionof amplification products are known to those skilled in the art. See,e.g., Sambrook et al. (2001) Molecular Cloninq: A Laboratory Manual(3^(rd) ed., Cold Spring Harbor Laboratory Press, NY) at 8.95; Livak etal. (1995) PCR Methods Appl. 4:357-362; and U.S. Pat. No. 5,538,848 andHeid et al. (1996) Genome Res. 6:986-994 (discussing TAQMAN® probes).

In certain embodiments, real-time PCR is conducted in the presence oftwo probes that selectively hybridize to adjacent regions of a strand ofthe amplification product. In certain such embodiments, the 3′ end ofthe first probe is attached to a donor fluorophore. The 5′ end of thesecond probe is attached to an acceptor fluorophore that is capable offluorescing at a different wavelength than the donor fluorophore.(Alternatively, in certain embodiments, the 3′ end of the first probe isattached to an acceptor fluorophore and the 5′ end of the second probeis attached to a donor fluorophore.) When the probes are hybridized to astrand of the amplification product, the 3′ end of the first probe is insufficient proximity to the 5′ end of the second probe, such that thefluorescence energy from the donor fluorophore is transferred to theacceptor fluorophore via FRET. Accordingly, an increase in fluorescencefrom the acceptor fluorophore indicates the presence of amplificationproducts.

In certain embodiments, real-time PCR is conducted in the presence of ahybridization-dependent probe. In certain embodiments, ahybridization-dependent probe is a hairpin probe, such as a “molecularbeacon.” See, e.g., U.S. Pat. Nos. 5,118,801; 5,312,728; and 5,925,517.In certain such embodiments, an oligonucleotide capable of forming ahairpin (stem-loop) structure is linked to a fluorophore at one end ofthe stem and a quencher moiety at the other end of the stem. Thequencher moiety quenches the fluorescence from the fluorophore when theoligonucleotide is in a hairpin configuration. The sequence of thehairpin loop is capable of selectively hybridizing to a strand of theamplification product. When such hybridization takes place, the hairpinconfiguration is disrupted, separating the fluorophore from the quenchermoiety. Accordingly, fluorescence from the fluorophore is increased.Thus, an increase in fluorescence indicates the presence ofamplification product.

Other hybridization-dependent probes include, but are not limited to,ECLIPSE™ probes (see, e.g., Afonina et al. (2002) Biotechniques32:940-44, 946-49). Certain quenching moieties for use withhybridization-dependent probes include, but are not limited to, Dabcyl,QSY7, QSY9, QSY22, and QSY35 (commercially available from MolecularProbes, Eugene, Oreg.).

In certain embodiments, real-time PCR is conducted using at least oneprimer comprising a 5′ portion that is not complementary to the targetnucleic acid. In certain such embodiments, the 5′ portion is capable offorming a hairpin (stem-loop) structure that is linked to a fluorophoreat one end of the stem and a quencher moiety at the other end of thestem. The quencher moiety quenches the fluorescence from the fluorophorewhen the 5′ portion is in a hairpin conformation. When the primerbecomes incorporated into a double-stranded amplification product, thehairpin conformation is disrupted. Accordingly, fluorescence from thefluorophore is increased. Thus, an increase in fluorescence indicatesthe presence of amplification product. Certain quenching moieties foruse with such primers include, but are not limited to, Dabcyl, QSY7,QSY9, QSY22, and QSY35 (commercially available from Molecular Probes).Certain fluorophores for use with such primers include, but are notlimited to, 6-FAM. An example of such a primer is a UNIPRIMER™ (ChemiconInternational Inc., Temecula, Calif.) or a SCORPION® primer (see, e.g.,Whitcombe et al. (1999) Nat. Biotechnol. 17:804-807).

4. Certain Hot-Start PCR

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a thermostable DNA polymerase is used in “hotstart” PCR. In certain embodiments known to those skilled in the art,“hot start” PCR is used to suppress non-specific binding of primer totemplate. See, e.g., Sambrook et al. (2001) Molecular Cloning: ALaboratory Manual (3^(rd) ed., Cold Spring Harbor Laboratory Press, NY)at 8.110 (describing “hot start” PCR). In certain embodiments of “hotstart” PCR, one or more components to be used in a PCR are preventedfrom functioning in the PCR until the reaction mixture reaches orexceeds a temperature at which non-specific priming does not occur or issubstantially reduced. Id.

In certain embodiments of “hot start” PCR, a thermostable DNA polymeraseis reversibly inactivated until a suitable temperature is reached. Forexample, in certain embodiments, an antibody to a thermostable DNApolymerase is used to reversibly block polymerase activity until asuitable temperature is reached. See, e.g., Kellogg et al. (1994)Biotechniques 16:1134-1137 (describing the use of antibodies to Taq DNApolymerase). In certain embodiments, a thermostable DNA polymerase ispartially or completely inactivated by a reversible chemicalmodification. In certain such embodiments, the chemical modification isreversed at a suitable temperature under amplification conditions. See,e.g., U.S. Pat. Nos. 5,773,258; 5,677,152; and 6,183,998. In certainembodiments, a thermostable DNA polymerase is inhibited by the bindingof a nucleic acid, such as an oligonucleotide, which dissociates fromthe thermostable DNA polymerase at a suitable temperature. See, e.g.,U.S. Pat. Nos. 6,183,967; 6,020,130; 5,874,557; 5,763,173; and5,693,502.

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a thermostable DNA polymerase is used in “hotstart” PCR. In certain such embodiments, an antibody to the nucleic acidbinding polypeptide is used to reversibly block nucleic acid bindingactivity and/or polymerase activity until a suitable temperature isreached.

In certain embodiments of “hot start” PCR, the thermostable DNApolymerase comprises a “cold-sensitive” mutant of a thermostable DNApolymerase. In certain such embodiments, the cold-sensitive mutant lackssubstantial activity until the reaction mixture reaches or exceeds atemperature at which non-specific priming does not occur or issubstantially reduced. Certain exemplary cold-sensitive mutants ofKlentaq235, Klentaq278, and naturally occurring Taq are known to thoseskilled in the art. For example, the W706R, E708D, E626K, and I707Lmutations confer cold sensitivity to Klentaq235, Klentaq278, ornaturally occurring Taq. See, e.g., Kermekchiev et al. (2003) NucleicAcids Res. 31:6139-6147; U.S. Pat. Nos. 6,333,159, 6,316,202, and6,214,557; and “Cesium Taq” (commercially available from DNA PolymeraseTechnology, Inc., St. Louis, Mo.).

5. Certain RT-PCR (Reverse Transcriptase-PCR)

RT-PCR is a modification of PCR in which an RNA template is firstreverse transcribed into its DNA complement or cDNA, followed byamplification of the resulting DNA using PCR. In certain embodiments,the reverse transcription reaction and the PCR reaction are carried outwith the same reaction mixture. In certain embodiments, the reversetranscription reaction and the PCR reaction proceed in differentreaction mixtures.

In certain embodiments in which two separate reaction mixtures areemployed, the RNA template is included with appropriate reagents,including a reverse transcriptase, for the reverse transcriptionreaction. In certain embodiments, the reverse transcription reactionproceeds for 30 minutes. In certain embodiments, the reversetranscription reaction proceeds at 60° C. One skilled in the art canalter times and temperatures as appropriate for various reversetranscriptase reactions. In certain two reaction mixture RT-PCRprocedures, a DNA polymerase is then added and PCR is carried out toamplify the cDNA produced in the reverse transcription reaction. Incertain two reaction mixture RT-PCR procedures, after the reversetranscription reaction, the cDNA from the reverse transcription reactionis separated out from the rest of the components in the mixture. ThatcDNA is then included in a second reaction mixture that includesreagents appropriate for amplifying the cDNA, including DNA polymerase,in a PCR reaction.

In certain embodiments, the reverse transcription reaction and the PCRreaction proceed in the same reaction mixture using an enzyme that canserve as both a reverse transcriptase and a DNA polymerase. In certainsuch embodiments, the reaction mixture including the RNA template areheld at an appropriate temperature for an appropriate period of time forthe reverse transcription reaction to generate cDNA, and then the PCRcycling is performed to amplify the cDNA. Certain exemplary polymerasesthat have both reverse transcriptase activity and polymerase activityare discussed in the application, including, but not limited to, thefollowing exemplary Family A DNA polymerases: Tth polymerase fromThermus thermophilus; Taq polymerase from Thermus aquaticus; Thermusthermophilus Rt4l A; Dictyoglomus thermophilum RT46B.1;Caldicellulosiruptor saccharolyticus Tok7B.1; Caldicellulosiruptor spp.Tok13B. 1; Caldicellulosiruptor spp. Rt69B.1; Clostridiumthermosulfurogenes; Thermotoga neapolitana; Bacillus caldolyticus EA1.3;Clostridium stercorarium; and Caldibacillus cellulovorans CA2. Certainexemplary polymerases that have both reverse transcriptase activity andpolymerase activity discussed in the application, include, but are notlimited to, a family B DNA polymerase that comprises one or moremutations that allow the polymerase to perform DNA polymerization usinga primed RNA template, such as Pfu DNA polymerase, with a point mutationL408Y or L408F (leucine to tyrosine or to phenylalane) in the conservedLYP motif. Certain exemplary fusion proteins are discussed in thisapplication that comprise a nucleic acid binding protein and a given DNApolymerase that can be used for RNA-templated DNA synthesis when thegiven DNA polymerase alone cannot perform DNA polymerization using aprimed RNA template. In certain such embodiments, the DNA polymerase inthe fusion protein is a Family B polymerase.

In certain embodiments, in which the reverse transcription reaction andthe PCR reaction proceed in the same reaction mixture, wax beadscontaining DNA polymerase for the PCR reaction are included in theinitial reaction mixture for the reverse transcription reaction. Afterthe reverse transcription reaction, the temperature is raised to meltthe wax to release the DNA polymerase for the PCR reaction.

In certain embodiments, RT-PCR is used to diagnose genetic disease ordetect RNA such as viral RNA in a sample. In certain embodiments, RT-PCRis used to determine the abundance of specific RNA molecules within acell or tissue as a measure of gene expression.

In certain embodiments, a fusion protein comprising a nucleic acidbinding protein and a polypeptide with reverse transcriptase activitycan be used to shorten the period of time for the reverse transcriptionreaction. For example, in certain embodiments, a fusion proteingenerates sufficient cDNA in a reverse transcription reaction thatproceeds for three to thirty (and all times between those endpoints)minutes.

In certain embodiments, a fusion protein stabilizes the primer-RNAtemplate duplex, thereby increasing the Tm of the primers above thepredicted Tm. Accordingly, in certain embodiments, the reversetranscription reaction is carried out at a temperature that is greaterthan the predicted Tm of the primers.

In certain embodiments, the reverse transcription reaction is carriedout at a temperature that is from about 1° C. to about 40° C. above thepredicted Tm of at least one of the primers (including all pointsbetween those endpoints). In certain such embodiments, the reversetranscription reaction is carried out at about 5° C., 10° C., 15° C., or20° C. above the predicted Tm of at least one of the primers.

In certain embodiments, the reverse transcription reaction is carriedout at any temperature from about 55° C. up to about 80° C. (includingall points between those endpoints). In certain such embodiments, thereverse transcription reaction is carried out at any temperature fromabout 62° C. to about 78° C.; from about 62° C. to about 75° C.; fromabout 65° C. to about 72° C.; from about 65° C. to about 75° C; fromabout 68° C. to about 72° C.; and from about 68° C. to about 75° C.

In certain embodiments, reverse transcription reaction at temperatureshigher than the reverse transcription reaction temperatures typicallyused in RT-PCR may, under certain circumstances, have beneficialeffects. For example, in certain embodiments, reverse transcriptionreaction at higher temperatures may improve primer specificity (i.e.,may alleviate “mispriming”). In certain embodiments, reversetranscription reaction at higher temperatures may allow for moreefficient amplification of problematic targets, such as targets havingrepetitive sequences or targets having complex secondary structure, suchas GC-rich targets.

6. Certain Nucleic Acid Sequencing

In certain embodiments, a fusion protein comprising a nucleic acidbinding polypeptide and a polymerase is used in a sequencing reaction.In certain embodiments, the sequencing reaction is a “cycle sequencing”reaction. See Sambrook et al. (2001) Molecular Cloning: A LaboratoryManual (3^(rd) ed., Cold Spring Harbor Laboratory Press, NY) at12.51-12.60, 12.94-12.114. In certain such embodiments, a nucleic acidtemplate is subjected to linear amplification using a single primer,thus generating single-stranded amplification products. In certainembodiments, the amplification is conducted in the presence of “chainterminators,” e.g., dideoxynucleotides. In certain embodiments, theprimer is labeled, e.g., with a radioisotope or fluorescent dye, toallow detection of chain-terminated amplification products. In certainembodiments, the chain terminator is labeled to allow detection ofchain-terminated amplification products. Exemplary chain terminatorsinclude, but are not limited to, radiolabeled dideoxynucleotideterminators and fluorescently labeled terminators, such as AppliedBiosystems' BigDye™ terminators (Applied Biosystems, Foster City,Calif.). In certain embodiments, cycle sequencing may employ any of thePCR cycling conditions described above, with the exception that only oneprimer is used, instead of at least two primers. In certain embodiments,amplification products are analyzed using an ABI PRISM® 310, 3100, or3100-Avant Genetic Analyzer, or an Applied Biosystems 3730 or 3730xI DNAAnalyzer (Applied Biosystems, Foster City, Calif.).

H. Certain Kits

In certain embodiments, a kit comprises any one or more of the nucleicacid binding polypeptides described above. In certain embodiments, a kitfurther comprises a nucleic acid modification enzyme. In certain suchembodiments the nucleic acid modification enzyme is a DNA polymerase. Incertain such embodiments, the DNA polymerase is a thermostable DNApolymerase. In certain such embodiments the nucleic acid modificationenzyme is a reverse transcriptase. In certain embodiments, a kit furthercomprises deoxynucleotides. In certain embodiments, a kit furthercomprises dideoxynucleotides.

In various embodiments, kits are provided. In certain embodiments, a kitcomprises any one or more fusion proteins comprising a nucleic acidbinding polypeptide and a polymerase. In certain such embodiments, thefusion protein comprises a nucleic acid binding polypeptide and athermostable DNA polymerase. In certain embodiments, a kit comprises anyone or more fusion proteins comprising a nucleic acid bindingpolypeptide and a reverse transcriptase. In certain embodiments, a kitfurther comprises deoxynucleotides. In certain embodiments, a kitfurther comprises dideoxynucleotides. In certain such embodiments, a kitfurther comprises fluorescently labeled dideoxynucleotides. In certainembodiments, a kit further comprises primers. In certain embodiments, akit further comprises one or more primers and/or probes for thedetection of amplification products. In certain such embodiments, a kitfurther comprises a 5′ nuclease probe or a hairpin probe. In certainembodiments, a kit further comprises a fluorescent indicator, such as anucleic acid binding dye.

VI. EXAMPLES

A. Cloning and Expression of Polynucleotides Encoding Nucleic AcidBinding Polypeptides

A polynucleotide encoding SEQ ID NO:1 was constructed by ligating thefollowing oligonucleotides (SEQ ID NOs:8-10) end-to,-end, such that the5′ end of SEQ ID NO:9 was ligated to the 3′ end of SEQ ID NO:8, and the5′ end of SEQ ID NO:10 was ligated to the 3′ end of SEQ ID NO:9. SEQ IDNO:8 5′ atgtccaaga agcagaaact Gaagttctac gacatTaagg cgaagcaggc gtttgag3′ SEQ ID NO:9 5′ acCgaccagt acgaggttat tgagaagcag acCgcccgcg gtccgatgatgttcgcc 3′ SEQ ID NO:10 5′ gtggccaaat cgccgtacac cggcatTaaa gtGtacCgCctgttaggcaa gaagaaataa 3′The capital letters in SEQ ID NOs:8-10 represent changes from thenaturally occurring PAE3192 sequence (SEQ ID NO:2). Those changes weremade to generate codons more favorable for the expression of SEQ ID NO:1in E. coli. Those changes do not result in any alterations in the aminoacid sequence of SEQ ID NO:1.

To ligate SEQ ID NOs:8-10 together, the following oligonucleotides (SEQID NOS:11-12) were first annealed to SEQ ID NOs:8-10 as discussed below.5′ gtactggtcg gtctcaaacg cctg 3′ SEQ ID NO:11 5′ cgatttggcc acggcgaacatcat 3′ SEQ ID NO:12SEQ ID NO:11 is complementary to the 3′ end of SEQ ID NO:8 and the 5′end of SEQ ID NO:9. Thus, the annealing of SEQ ID NO:11 to SEQ IDNOs:8-9 created a region of double-stranded DNA where SEQ ID NO:11 spansthe junction of SEQ ID NOS:8-9. This region of double-stranded DNA was asuitable substrate for DNA ligase. Likewise, SEQ ID NO:12 iscomplementary to the 3′ end of SEQ ID NO:9 and the 5′ end of SEQ IDNO:10. Thus, the annealing of SEQ ID NO:12 to SEQ ID NOS:9-10 created aregion of double-stranded DNA where SEQ ID NO:12 spans the junction ofSEQ ID NOS:9-10.

SEQ ID NOs:8-10 were then ligated. The resulting polynucleotide (SEQ IDNO:13) was amplified by PCR.

A polynucleotide encoding SEQ ID NO:6 was constructed by ligating thefollowing oligonucleotides (SEQ ID NOs:14-16) end-to-end: SEQ ID NO:145′ atgccGaaga aggagaagat Taagttcttc gacctGgtcg ccaagaagta ctacgag 3′ SEQID NO:15 5′ actgacaact acgaagtcga gatTaaggag actaagCgCg gcaagtttCgCttcgcc 3′ SEQ ID NO:16 5′ aaagccaaga gcccgtacac cggcaagatc ttctatCgCgtgctGggcaa agcctag 3′The capital letters represent changes from the naturally occurringAPE3192 sequence (SEQ ID NO:7). Those changes were made to generatecodons more favorable for the expression of SEQ ID NO:6 in E. coli.Those changes do not result in any alterations in the amino acidsequence of SEQ ID NO:6.

The following oligonucleotides (SEQ ID NOs:17-18) were annealed to SEQID NOs:14-16 to create regions of double-stranded DNA spanning thejunctions between SEQ ID NOs:14-15 and SEQ ID NOs:15-16. 5′ gtagttgtcagtctcgtagt actt 3′ SEQ ID NO:17 5′ gctcttggct ttggcgaagc gaaa 3′ SEQ IDNO:18SEQ ID NOs:14-16 were then ligated. The resulting polynucleotide (SEQ IDNO:19) was amplified by PCR.

SEQ ID NO:13 was cloned into the pET16b vector (Novagen, Milwaukee,Wis.) using standard recombinant methods. That vector allows expressionof the cloned sequences from the inducible T7 promoter. It also includessequences encoding polyhistidine (10×His) followed by a Factor Xacleavage site upstream of the cloning site. Thus, the encoded proteinsare tagged at their N-termini with a polyhistidine moiety. Recombinantvector comprising SEQ ID NO:13 was transformed into competent E. colihost cells using standard methods.

SEQ ID NO:19 was also cloned into the pET16b vector using standardrecombinant methods. Recombinant vector comprising SEQ ID NO:19 wastransformed into competent E. coli host cells using standard methods.

Host cells containing a recombinant vector comprising SEQ ID NO:13 areinduced to express a tagged polypeptide comprising SEQ ID NO:1 by addingIPTG to the media in which the host cells are grown. The taggedpolypeptide is isolated from the host cells by affinity chromatographyusing nickel-NTA resin. In certain embodiments, the polyhistidine tag isremoved from the isolated polypeptide by treatment with Factor Xa.

Host cells containing a recombinant vector comprising SEQ ID NO:19 areinduced to express a tagged polypeptide comprising SEQ ID NO:6 by addingIPTG to the media in which the host cells are grown. The taggedpolypeptide is isolated from the host cells by affinity chromatographyusing nickel-NTA resin. In certain embodiments, the polyhistidine tag isremoved from the isolated polypeptide by treatment with Factor Xa.

B. Assay for Stabilization of a DNA Duplex from Thermal Denaturation

The ability of a nucleic acid binding polypeptide to stabilize a DNAduplex from thermal denaturation is demonstrated by the following assay,which measures the increase in the Tm of a nucleic acid in the presenceof a nucleic acid binding polypeptide. See, e.g., Baumann et al. (1994)Nature Struct. Biol. 1:808-819; and McAfee et al. (1995) Biochem.34:10063-10077. Poly(dl-dC) at a concentration of about 70 μM (innucleotides) is combined with a nucleic acid binding polypeptide at aconcentration of about 350 μM in 5 mM Tris·Cl (pH 7.0). Poly(dl-dC) at aconcentration of about 70 μM (in nucleotides) in 5 mM Tris·Cl (pH 7.0)without a nucleic acid binding polypeptide is used as a negativecontrol. The absorbance of the poly(dl-dC) with and without a nucleicacid binding polypeptide is measured at 260 nm as a function oftemperature using a spectrophotometer. The temperature is increased insteps, and absorbance is measured at each step. For each step, thetemperature is raised by 1° C. over 30 seconds, followed by a holdingtime of 60 seconds prior to the measuring of absorbance. A melting curveis generated based on the increase in absorbance as a function oftemperature. The Tm (temperature at which 50% of the poly(dl-dC) isdenatured) occurs at the inflection point of the melting curve. The Tmof poly(dl-dC) in the negative control is subtracted from the Tm ofpoly(dl-dC) in the presence of a nucleic acid binding polypeptide todetermine the increase in Tm due to the presence of the nucleic acidbinding polypeptide.

The experiment discussed in Example K(2) below can be used to test theability of a nucleic acid binding polypeptide to stabilize a DNA:RNAduplex from thermal denaturation.

C. Construction and Expression of Fusion Proteins Comprising a NucleicAcid Binding Polypeptide and a Thermostable DNA Polymerase

1. Fusion Proteins Comprising Pfu DNA Polymerase

a) Fusion Proteins Comprising Pfu and Pae3192

A fusion protein comprising Pae3192 (SEQ ID NO:1) joined to theC-terminus of full length Pfu DNA polymerase was constructed as follows.An NdeI-XhoI restriction fragment comprising a polynucleotide sequenceencoding full length Pfu DNA polymerase in frame with a polynucleotidesequence encoding Pae3192 (SEQ ID NO:13) was cloned into the NdeI andXhoI sites of the pET16b vector (Novagen, Milwaukee, Wis.) usingstandard recombinant methods. The resulting recombinant vector (pDS2r)encodes a fusion protein comprising Pae3192 joined to the C-terminus ofPfu DNA polymerase by a Gly-Thr-Gly-Gly-Gly-Gly peptide linker. A 10×Hisaffinity tag is present at the N-terminus of the fusion protein. Thefusion protein, designated “10His-Pfu-Pae3192,” has the amino acidsequence shown in SEQ ID NO:23. The polynucleotide sequence encoding10His-Pfu-Pae3192 is shown in SEQ ID NO:22.

The recombinant vector pDS2r was transformed into competent E. coli hostcells. Host cells comprising pDS2r were induced to express10His-Pfu-Pae3192 by adding IPTG to the media in which the host cellswere grown. 10His-Pfu-Pae3192 was isolated from the host cells byaffinity chromatography using nickel-NTA resin.

In certain embodiments, the polyhistidine tag is removed from10His-Pfu-Pae3192 by treatment with Factor Xa to yield the fusionprotein shown in SEQ ID NO:24. That fusion protein is designated“Pfu-Pae3192.”

b) Fusion Proteins Comprising Pfu and Ape3192

A fusion protein comprising Ape3192 (SEQ ID NO:6) joined to theC-terminus of full length Pfu DNA polymerase was constructed as follows:An NdeI-XhoI restriction fragment comprising a polynucleotide sequenceencoding full length Pfu DNA polymerase in frame with a polynucleotidesequence encoding Ape3192 (SEQ ID NO:19) was cloned into the NdeI andXhoI sites of the pET16b vector using standard recombinant methods. Theresulting recombinant vector (pDS1r) encodes a fusion protein comprisingApe3192 joined to the C-terminus of Pfu DNA polymerase by aGly-Thr-Gly-Gly-Gly-Gly peptide linker. A 10×His affinity tag is presentat the N-terminus of the fusion protein. The fusion protein, designated“10His-Pfu-Ape3192,” has the amino acid sequence shown in SEQ ID NO:26.The polynucleotide sequence encoding 10His-Pfu-Ape3192 is shown in SEQID NO:25.

The recombinant vector pDS1r was transformed into competent E. coli hostcells. Host cells comprising pDS1r were induced to express10His-Pfu-Ape3192 by adding IPTG to the media in which the host cellswere grown. 10His-Pfu-Ape3192 was isolated from the host cells byaffinity chromatography using nickel-NTA resin.

In certain embodiments, the polyhistidine tag is removed from10His-Pfu-Ape3192 by treatment with Factor Xa to yield the fusionprotein shown in SEQ ID NO:27. That fusion protein is designated“Pfu-Ape3192.”

c) Fusion Proteins Comprising Pfu and Sso7d

A fusion protein comprising Sso7d (SEQ ID NO:20 lacking the firstmethionine) joined to the C-terminus of full length Pfu DNA polymerasewas constructed as follows: An NdeI-XhoI restriction fragment comprisinga polynucleotide sequence encoding full length Pfu DNA polymerase inframe with a polynucleotide sequence encoding Sso7d was cloned into theNdeI and XhoI sites of the pET16b vector using standard recombinantmethods. The resulting recombinant vector (pDS3r) encodes a fusionprotein comprising Sso7d joined to the C-terminus of Pfu DNA polymeraseby a Gly-Thr-Gly-Gly-Gly-Gly peptide linker. A 10×His affinity tag ispresent at the N-terminus of the fusion protein. The fusion protein,designated “10His-Pfu-Sso7d,” has the amino acid sequence shown in SEQID NO:49. The polynucleotide sequence encoding 10His-Pfu-Sso7d is shownin SEQ ID NO:51.

The recombinant vector pDS3r was transformed into competent E. coli hostcells. Host cells comprising pDS3r were induced to express10His-Pfu-Sso7d by adding IPTG to the media in which the host cells weregrown. 10His-Pfu-Sso7d was isolated from the host cells by affinitychromatography using nickel-NTA resin.

In certain embodiments, the polyhistidine tag is removed from10His-Pfu-Sso7d by treatment with Factor Xa to yield the fusion proteinshown in SEQ ID NO:50. That fusion protein is designated “Pfu-Sso7d.”

d) Fusion Proteins Comprising Pfu and Pae3192

A fusion protein comprising Pae3192 (SEQ ID NO:1)joined to theC-terminus of full length Pfu DNA polymerase with two mutations D141Aand E143A was constructed. The fusion protein was constructed using thesame methods described in Example C(1)(a) above, except thepolynucleotide sequence encoded full length Pfu DNA polymerase with analanine at position 141 of Pfu DNA polymerase rather than aspartic acidand with an alanine at position 143 of Pfu DNA polymerase rather thanglutamic acid. The fusion protein, designated 10His-Pfu-Pae3192,exo-minus version” has the amino acid sequence shown in SEQ ID NO:23,except the aspartic acid at position 141 is replaced with alanine andthe glutamic acid at position 143 is replaced with alanine.

2. Fusion Proteins Comprising Taq DNA Polymerase

a) Fusion Proteins Comprising Pae3192 and Taq DNA Polymerase

A fusion protein comprising Pae3192 (SEQ ID NO:1)joined to theN-terminus of Taq DNA polymerase (SEQ ID NO:31 lacking the first twoamino acid residues) was constructed as follows. A polynucleotideencoding Pae3192 (SEQ ID NO:13) was cloned in frame at the 5′ end of apolynucleotide encoding Taq DNA polymerase in the pET16b vector. Theresulting recombinant vector (pDS17-7) encodes a fusion proteincomprising Pae3192 joined to the N-terminus of Taq DNA polymerase by aGly-Gly-Val-Thr-Ser peptide linker. A 10×His affinity tag is present atthe N-terminus of the fusion protein. The fusion protein, designated“10His-Pae3192-Taq,” has the amino acid sequence shown in SEQ ID NO:33.The polynucleotide sequence encoding 10His-Pae3192-Taq is shown in SEQID NO:32. The recombinant vector pDS1 7-7 was transformed into competenthost cells.

Expression of 10His-Pae3192-Taq is induced in the host cells using IPTG.10His-Pae3192-Taq is isolated from the host cells by affinitychromatography using nickel-NTA resin. In certain embodiments, thepolyhistidine tag is removed from 10His-Pae3192-Taq by treatment withFactor Xa to yield a fusion protein having the amino acid sequence shownin SEQ ID NO:34. That fusion protein is designated “Pae3192-Taq.”

b) Fusion Proteins Comprising Ape3192 and Taq DNA Polymerase

A fusion protein comprising Ape3192 (SEQ ID NO:6) joined to theN-terminus of Taq DNA polymerase (SEQ ID NO:31 lacking the first twoamino acid residues) was constructed as follows. A polynucleotideencoding Ape3192 (SEQ ID NO:19) was cloned in frame at the 5′ end of apolynucleotide encoding Taq DNA polymerase in the pET16b vector. Theresulting recombinant vector (pDS16-3) encodes a fusion proteincomprising Ape3l92 joined to the N-terminus of Taq DNA polymerase by aGly-Gly-Val-Thr-Ser peptide linker. A 10×His affinity tag is present atthe N-terminus of the fusion protein. The fusion protein, designated“10His-Ape3192-Taq,” has the amino acid sequence shown in SEQ ID NO:36.The polynucleotide sequence encoding 10His-Ape3192-Taq is shown in SEQID NO:35. The recombinant vector pDS16-3 was transformed into competenthost cells.

Expression of 10His-Ape3192-Taq is induced in the host cells using IPTG.10His-Ape3192-Taq is isolated from the host cells by affinitychromatography using nickel-NTA resin. In certain embodiments, thepolyhistidine tag is removed from 10His-Ape3192-Taq by treatment withFactor Xa to yield the fusion protein shown in SEQ ID NO:37. That fusionprotein is designated “Ape3192-Taq.”

c) Fusion Proteins Comprising Pae3192 and the Stoffel Fragment

A fusion protein comprising Pae3192 (SEQ ID NO:1) joined to theN-terminus of a Stoffel fragment of Taq DNA polymerase (amino acidresidues 291-832 of SEQ ID NO:31) was constructed as follows. Apolynucleotide encoding Pae3192 (SEQ ID NO:13) was cloned in frame atthe 5′ end of a polynucleotide encoding the Stoffel fragment in thepET16b vector. The resulting recombinant vector (pDS25-7) encodes afusion protein comprising Pae3192 joined to the N-terminus of theStoffel fragment by a Gly-Gly-Val-Thr-Ser peptide linker. A 10×Hisaffinity tag is present at the N-terminus of the fusion protein. Thefusion protein, designated “10His-Pae3l92-Taq_(ST),” has the amino acidsequence shown in SEQ ID NO:39. The polynucleotide sequence encoding10His-Pae3192-Taq_(ST) is shown in SEQ ID NO:38. The recombinant vectorpDS25-7 was transformed into competent host cells.

Expression of 10His-Pae3192-Taq_(ST) is induced in the host cells usingIPTG. The fusion protein is isolated from the host cells by affinitychromatography using nickel-NTA resin. In certain embodiments, thepolyhistidine tag is removed from 10His-Pae3192-Taq_(ST) by treatmentwith Factor Xa to yield the fusion protein shown in SEQ ID NO:40. Thatfusion protein is designated “Pae3192-Taq_(ST).”

d) Fusion Proteins Comprising Ape3192 and the Stoffel Fragment

A fusion protein comprising Ape3192 (SEQ ID NO:6) joined to theN-terminus of a Stoffel fragment of Taq DNA polymerase (amino acidresidues 291-832 of SEQ ID NO:31) was constructed as follows. Apolynucleotide encoding Ape3192 (SEQ ID NO:19) was cloned in frame atthe 5′ end of a polynucleotide encoding the Stoffel fragment in thepET16b vector. The resulting recombinant vector (pDS24-4) encodes afusion protein comprising Ape3192 joined to the N-terminus of theStoffel fragment by a Gly-Gly-Val-Thr-Ser peptide linker. A 10×Hisaffinity tag is present at the N-terminus of the fusion protein. Thefusion protein, designated “10His-Ape3192-Taq_(ST),” has the amino acidsequence shown in SEQ ID NO:42. The polynucleotide sequence encoding10His-Ape3192-Taq_(ST) is shown in SEQ ID NO:41. The recombinant vectorpDS24-4 was transformed into competent host cells.

Expression of 10His-Ape3192-Taq_(ST) is induced in the host cells usingIPTG. The fusion protein is isolated from the host cells by affinitychromatography using nickel-NTA resin. In certain embodiments, thepolyhistidine tag is removed from 10His-Ape3192-Taq_(ST) by treatmentwith Factor Xa to yield the fusion protein shown in SEQ ID NO:43. Thatfusion protein is designated “Ape3192-Taq_(ST).”

D. Use of Fusion Proteins in “Fast” PCR

Fusion proteins were used in PCR reactions having rapid cycling times. Aset of five reaction mixtures were prepared as follows: Component (stockFinal concentration) Volume concentration Lambda (λ) DNA 2 μl 1 ng/μl(10 ng/μl) dNTPs (2.5 mM each) 2 μl 250 μM each Buffer (10× or 5×) 2 or4 μl 1× Forward primer (10 μM) 1 μl 0.5 μM Reverse primer (10 μM) 1 μl0.5 μM Enzyme 0.5 μl ˜1 Unit dH₂O 11.5 or 9.5 μl 20 μl final volume

All five reaction mixtures contained the following forward and reverseprimers: (SEQ ID NO:47) 5′-AGCCAAGGCCAATATCTAAGTAAC-3′ (Tm=54.1° C.)(SEQ ID NO:48) 5′-CGAAGCATTGGCCGTAAGTG-3′ (Tm=58.4° C.)

The reaction mixtures contained one of the following enzyme-buffercombinations, as indicated below: Reaction Buffer (stock mixture Enzymeconcentration) A Cloned Pfu polymerase 10× Cloned Pfu (Stratagene, LaJolla, CA) polymerase buffer (Stratagene) B 10His-Pfu-Ape3192 5× PhusionHF buffer (SEQ ID NO: 26) (Finnzymes, Espoo, Finland) C10His-Pfu-Pae3192 5× Phusion HF buffer (SEQ ID NO: 23) (Finnzymes) D10His-Pfu-Sso7d 5× Phusion HF buffer (SEQ ID NO: 49) (Finnzymes) EAmpliTaq (Roche 10× AmpliTaq buffer Molecular Systems, (Roche MolecularPleasanton, CA) Systems)

The reaction mixtures were subjected to “fast” PCR cycling conditionsusing an Applied Biosystems 9800 Fast Thermal Cycler (AppliedBiosystems, Foster City, Calif.), as follows: 98° C., 30 sec; 99° C., 1sec; and 65° C., 1 sec. 30 cycles

After the 30 cycles, the reaction mixtures were analyzed by agarose gelelectrophoresis. See FIG. 1, Set 1. Reaction mixtures A and E did notcontain detectable amplification product. See lanes A and E of FIG. 1,Set 1. Unexpectedly, reaction mixtures B, C, and D contained substantialamounts of amplification product having the predicted size. See lanes B,C, and D of FIG. 1, Set 1. (Size markers are shown in lane M.) Thus, thefusion proteins 10His-Pfu-Ape3192, 10His-Pfu-Pae3192, and10His-Pfu-Sso7d efficiently amplified lambda DNA under fast PCR cyclingconditions at an annealing temperature of 65° C., whereas thethermostable DNA polymerases Pfu and AmpliTaq did not.

An identical set of reaction mixtures were subjected to fast PCR cyclingconditions at a higher annealing/extension temperature, as follows: 98°C., 30 sec; 99° C., 2 sec; and 70° C., 2 sec. 30 cycles

After the 30 cycles, the reaction mixtures were analyzed by agarose gelelectrophoresis, shown in FIG. 1, Set 2. Reaction mixtures A and E didnot contain detectable amplification product. See lanes A and E of FIG.1, Set 2. Unexpectedly, reaction mixtures B, C, and D containedsubstantial amounts of amplification product having the predicted size.See lanes B, C, and D of FIG. 1, Set 2. Thus, the fusion proteins10His-Pfu-Ape3192, 10His-Pfu-Pae3192, and 10His-Pfu-Sso7d efficientlyamplified lambda DNA under fast PCR cycling conditions at an annealingtemperature of 70° C., whereas the thermostable DNA polymerases Pfu andAmpliTaq did not.

To investigate the effect of a polyhistidine tag on the performance offusion proteins, two reaction mixtures identical to reaction mixtures Band C above were prepared. A third reaction mixture “F” was prepared asdescribed for reaction mixtures B and C, except that the enzyme used inreaction mixture F was Pfu-Pae3192 (SEQ ID NO:24). Reaction mixtures B,C, and F were subjected to “fast” PCR cycling conditions using anApplied Biosystems 9800 Fast Thermal Cycler (Applied Biosystems, FosterCity, Calif.), as follows: 98° C., 30 sec; 99° C., 1 sec; and 65° C., 1sec. 30 cycles

After the 30 cycles, the reaction mixtures were analyzed by agarose gelelectrophoresis. All three reaction mixtures contained detectableamplification product. However, reaction mixture F had qualitativelyless amplification product than reaction mixtures B and C. Thus, thefusion proteins 10His-Pfu-Ape3192 and 10His-Pfu-Pae3192, which bothcontain a polyhistidine tag, amplified lambda DNA more efficiently underfast PCR cycling conditions than Pfu-Pae3192, which does not contain apolyhistidine tag.

E. Processivity Assay

The processivity of a DNA polymerase is compared to the processivity ofa fusion protein comprising a nucleic acid binding polypeptide and a DNApolymerase using a processivity assay based on that of Wang et al.(2004) Nuc. Acids Res. 32:1197-1207. A 5′ FAM-labeled primer of sequence5′ gttttcccagtcacgacgttgtaaaacgacggcc 3′ (SEQ ID NO:29) is added tosingle-stranded M13mp18 DNA in a reaction composition comprising 10 mMTris-HCl pH 8.8, 50 mM KCl, 2.5 mM MgCl₂, 250 μm dNTPs, and 0.1% TritonX-100. The concentrations of the primer and M13mp18 template are 50 nMand 80 nM, respectively. The primer is annealed to the single-strandedM13mp18 DNA template by heating the mixture to 90° C. for 5 minutes,cooling to 72° C. at 0.1° C. per second, incubating at 72° C. for 10minutes, and cooling to 4° C. at 0.1° C. per second.

Two parallel reactions are prepared. In the first reaction, athermostable DNA polymerase is added to a final concentration of about1:4000 (DNA polymerase:template) in 20 μl of the above reactioncomposition. In the second reaction, a fusion protein comprising athermostable DNA polymerase and a nucleic acid binding polypeptide isadded to a final concentration of about 1:4000 (fusion protein:template)in 20 μl of the above reaction composition.

DNA synthesis is initiated in the reactions by incubating them at 72° C.Samples from each reaction are taken at various time points. The samplesare diluted in gel loading dye, and the primer extension products in thesamples are analyzed by denaturing polyacrylamide gel electrophoresisusing an ABI 377 DNA Sequencer (Applied Biosystems, Foster City,Calif.). The median product length is determined based on theintegration of all detectable primer extension products. When the medianproduct length does not change with an increase in reaction time or adecrease in polymerase concentration (to ensure that the template is inexcess), that length is used as a measure of processivity.

F. Use of Nucleic Acid Binding Polypeptides to Increase Processivity ofa DNA Polymerase

The ability of a nucleic acid binding polypeptide to increase theprocessivity of a DNA polymerase is assessed using a processivity assaybased on that of Wang et al. (2004) Nuc. Acids Res. 32:1197-1207. A 5′FAM-labeled primer of sequence 5′ gttttcccagtcacgacgttgtaaaacgacggcc 3′(SEQ ID NO:29) is added to single stranded M13mp18 DNA in a reactioncomposition comprising 10 mM Tris-HCl pH 8.8, 50 mM KCl, 2.5 mM MgCl₂,250 μm dNTPs, and 0.1% Triton X-100. The concentrations of the primerand M13mp18 template are 50 nM and 80 nM, respectively. The primer isannealed to the single stranded M13mp18 DNA template by heating themixture to 90° C. for 5 minutes, cooling to 72° C. at 0.1° C. persecond, incubating at 72° C. for 10 minutes, and cooling to 4° C. at0.1° C. per second. A thermostable DNA polymerase, such as Taq DNApolymerase, is added to the above reaction composition at aconcentration of about 1:4000 (DNA polymerase:template).

Two parallel reactions are prepared. In one of the parallel reactions, anucleic acid binding polypeptide is added to a final concentration ofabout 70 μg/ml in 20 μl of the above reaction composition. The secondparallel reaction contains 20 μl of the above reaction composition withno added nucleic acid binding polypeptide.

DNA synthesis is initiated in the reaction compositions by incubatingthem at 72° C. Samples from each reaction are taken at various timepoints. The samples are diluted in gel loading dye, and the primerextension products in the samples are analyzed by denaturingpolyacrylamide gel electrophoresis using an ABI 377 DNA Sequencer. Themedian product length is determined based on the integration of alldetectable primer extension products. When the median product lengthdoes not change with an increase in reaction time or a decrease inpolymerase concentration (to ensure that the template is in excess),that length is used as a measure of processivity.

G. Use of Nucleic Acid Binding Polypeptides to Increase the Efficiency(Speed and Specificity) of a Hybridization-Based Detection Assay

1. Annealing Assay

The ability of a nucleic acid binding polypeptide to increase thespecificity of a hybridization-based detection assay is measured usingan annealing assay based on that of Guagliardi et al. (1997) J. Mol.Biol. 267:841-848. A first set of two reaction compositions is preparedas follows: In a first reaction composition, single stranded M13mp18circular DNA (0.05 pmol) is combined with an equal amount of ³²Pend-labeled oligonucleotide of sequence 5′-gtaaaacgacggccagt-3′ (SEQ IDNO:20) in a buffered reaction mixture (20 mM Tris-HCl pH 7.5, 2 mM DTT,5 mM MgCl2, 100 μg/ml BSA). In a second reaction composition, singlestranded M13mp18 circular DNA (0.05 pmol) is combined with an equalamount of ³²P end-labeled oligonucleotide of sequence5′-gtaaaacgtcggccagt-3′ (SEQ ID NO:21) in a buffered reaction mixture(20 mM Tris-HCl pH 7.5, 2 mM DTT, 5 mM MgCl2, 100 μg/ml BSA). Thenucleotide indicated in bold is a mismatch with respect to the M13mp18DNA sequence. A nucleic acid binding polypeptide is added separately toboth reaction compositions at a final concentration of about 5 μg/ml.

A second set of two reaction compositions is prepared. The second set isthe same as the first set of reaction compositions, except that anucleic acid binding polypeptide is not added to either the first orsecond reaction composition of the second set of reaction compositions.The final volume of each reaction composition is 10 μl.

The reaction compositions are incubated at 60° C. for three minutes. Thereactions are stopped by adding 1% SDS in standard loading dye to eachreaction composition. The reactions are analyzed by 1.5% agarose gelelectrophoresis followed by autoradiography to visualize annealedproduct, which can be distinguished from unannealed probe by its slowermobility. Annealed product is quantified for each reaction usingstandard densitometric methods. An increase in the amount of annealedproduct in the first reaction compared to the second reaction isdetermined for both sets of reactions. The ability of a nucleic acidbinding polypeptide to increase the specificity of hybridization isdemonstrated by a larger increase in the amount of annealed product forthe first set of reactions compared to the second set of reactions.

To test the annealing of RNA to DNA, the assay discussed above can beperformed by replacing the DNA sequences SEQ ID NO:20 and SEQ ID NO:21with their RNA sequence counterparts.

2. Microarray-Based Assay

The ability of a nucleic acid binding polypeptide to increase the speedand specificity of a hybridization-based detection assay is alsodemonstrated by a decrease in the hybridization time (approximately 16hours) required to perform a typical microarray-based detection assay. Atypical microarray-based detection assay may be performed, for example,using the Mouse Genome Survey Microarray system (Applied Biosystems,Foster City, Calif.; P/N 4345065). That system includes reagents,hybridization controls, and reference nucleic acids that can be used todetect selective hybridization of a reference nucleic acid to a probe(i.e., a portion of a mouse cDNA) immobilized on a microarray. In anexemplary assay, a nucleic acid binding polypeptide is added to thehybridization solution at a concentration of about 50 to 250 ug/mL. Thehybridization time is from about 1 to 30 minutes at a temperature ofabout 45° C. to 75° C. The arrays are washed, and hybridization isdetected using the Chemiluminescence Detection Kit (Applied Biosystems,Foster City, Calif., P/N 4342142) according to the manufacturer'sinstructions. The arrays are analyzed using the Applied Biosystems 1700Chemiluminescent Microarray Analyzer (Applied Biosystems, Foster City,Calif., P/N 4338036). To test hybridization of RNA to the DNA on amicroarray, one can use RNA as the reference nucleic acid.

H. Use of Fusion Proteins to Increase Processivity of Taq DNA Polymerase

The increase in processivity of a fusion protein comprising Taq DNApolymerase relative to Taq DNA polymerase alone is assessed using aprocessivity assay based on that of Wang et al. (2004) Nuc. Acids Res.32:1197-1207. A 5′ FAM-labeled primer of sequence 5′gttttcccagtcacgacgttgtaaaacgacggcc 3′ (SEQ ID NO:29) is added to singlestranded M13mp18 DNA in a mixture comprising 10 mM Tris-HCl pH 8.8, 50mM KCl, 2.5 mM MgCl₂, 250 μm dNTPs, and 0.1% Triton X-100. Theconcentrations of the primer and M13mp18 template are 50 nM and 80 nM,respectively. The primer is annealed to the single stranded M13mp18 DNAtemplate by heating the mixture to 90° C. for 5 minutes, cooling to 72°C. at 0.1° C. per second, incubating at 72° C. for 10 minutes, andcooling to 4° C. at 0.1° C. per second.

A reaction composition is prepared in which a fusion protein comprisingTaq DNA polymerase is added at a molar concentration of about 1:4000(fusion protein:template) to 20 μl of the above mixture. A controlreaction composition is prepared in which Taq DNA polymerase is added ata molar concentration of about 1:4000 (DNA polymerase:template) to 20 μlof the above mixture. DNA synthesis is initiated in the reactioncompositions by incubating them at 72° C. Samples from each reaction aretaken at various time points. The samples are diluted in gel loadingdye, and the primer extension products are analyzed by denaturingpolyacrylamide gel electrophoresis using an ABI 377 DNA Sequencer. Themedian product length is determined based on the integration of alldetectable primer extension products. When the median product lengthdoes not change with an increase in reaction time or a decrease inpolymerase concentration, that length is used as a measure ofprocessivity.

I. Use of Fusion Proteins to Increase Processivity of Pfu DNA Polymerase

The increase in processivity of a fusion protein comprising Pfu DNApolymerase relative to Pfu DNA polymerase alone is assessed using aprocessivity assay based on that of Wang et al. (2004) Nuc. Acids Res.32:1197-1207. A 5′ FAM-labeled primer of sequence 5′gttttcccagtcacgacgttgtaaaacgacggcc 3′ (SEQ ID NO:29) is added to singlestranded M13mp18 DNA in a mixture comprising 10 mM Tris-HCl pH 8.8, 50mM KCl, 2.5 mM MgCl₂, 250 μm dNTPs, and 0.1% Triton X-100. Theconcentrations of the primer and M13mp18 template are 50 nM and 80 nM,respectively. The primer is annealed to the single stranded M13mp18 DNAtemplate by heating the mixture to 90° C. for 5 minutes, cooling to 72°C. at 0.1° C. per second, incubating at 72° C. for 10 minutes, andcooling to 4° C. at 0.1° C. per second.

A reaction composition is prepared in which a fusion protein comprisingPfu DNA polymerase is added at a molar concentration of about 1:4000(fusion protein:template) to 20 μl of the above mixture. A controlreaction composition is prepared in which Pfu DNA polymerase is added ata molar concentration of about 1:4000 (DNA polymerase:template) to 20 μlof the above mixture. DNA synthesis is initiated in the reactioncompositions by incubating them at 72° C. Samples from each reaction aretaken at various time points. The samples are diluted in gel loadingdye, and the primer extension products are analyzed by denaturingpolyacrylamide gel electrophoresis using an ABI 377 DNA Sequencer. Themedian product length is determined based on the integration of alldetectable primer extension products. When the median product lengthdoes not change with an increase in reaction time or a decrease inpolymerase concentration, that length is used as a measure ofprocessivity.

One skilled in the art will readily recognize that the above assay maybe modified so as to assess the processivity of a fusion proteincomprising a DNA polymerase other than Taq or Pfu.

J. Use of Fusion Proteins in PCR

The ability of a fusion protein comprising a nucleic acid bindingpolypeptide and a thermostable DNA polymerase (e.g., Taq or Pfu) toincrease the efficiency of PCR is demonstrated using a typical PCRreaction. An exemplary PCR reaction is prepared which contains PCRbuffer (1×), dNTPs (200 μM each), template DNA (250 ng), forward andreverse primers (0.25 μM each) and fusion protein (about 0.5 to 2.5units) in a final volume of 50 μl. As a control reaction, thermostableDNA polymerase alone is used in place of the fusion protein. The primersused in the PCR reaction are tPAF7(5′-ggaagtacagctcagagttctgcagcacccctgc-3′ (SEQ ID NO:45)) and tPAR10(5′-gatgcgaaactgaggctggctgtactgtctc-3′ (SEQ ID NO:46)). The template DNAis human genomic DNA (Roche, Indianapolis, Ind., P/N 1-691-112). Theprimers tPAF7 and tPAR10 amplify a product of approximately 5 kb fromhuman genomic DNA. If the fusion protein being used in the PCR reactioncomprises Pfu DNA polymerase, then the standard PCR buffer for Pfu(Stratagene; La Jolla, Calif.) is used, except that the KClconcentration is elevated. The final working concentration (1×) of thebuffer thus contains 20 mM Tris, pH 8.8; 10 mM (NH₄)₂SO₄, 0.1% TritonX-100, 2 mM MgSO₄, 100 μg/mL BSA and 60 mM KCl. If the fusion proteinbeing used in the PCR reaction comprises Taq DNA polymerase, thestandard PCR buffer for Taq (Applied Biosystems, Foster City, Calif.) isused. Cycling is performed as follows: initial dentaturation (98° C., 30sec); denaturation (98° C., 10 sec); annealing (65° C., 10 sec); and{close oversize bracket} 29 cycles extension (72° C., 2 min); and finalextension (72° C., 10 min).

An aliquot of the reaction is analyzed by agarose gel electrophoresisalong with an appropriate size standard, stained with ethidium bromide,and then visualized by fluorescence.

K. Pae3192 Binding to DNA:DNA Duplexes and DNA:RNA Duplexes

The ability of Pae3192 to bind to DNA:DNA duplexes and DNA:RNA duplexeswas tested.

1. Gel-Shift Experiments

Gel shift analysis is an accepted way to assay binding of a polypeptideto a nucleic acid (see, for example, Kamashev et al., EMBO J.,19(23):6527-6535 (2000). Binding of Sso7d to DNA has been shown usinggel-shift assays (see, for example, Guagliardi et al., J. Mol. Biol.,267(4):841-848 (1997).

Gel-shift experiments were carried out using 150 nM 42-mer duplex andseparate experiments were performed with 0, 1.5, 3, 6 or 12 uM Pae3192protein. A DNA:DNA duplex was created by annealing DNA oligonucleotides1a and 2a of Table 1 below. An RNA:RNA duplex was created by annealingRNA oligonucleotides 1b and 2b of Table 1 below. A DNA:RNA duplex wascreated by annealing DNA oligonucleotide 1a to RNA oligonucleotide 2b ofTable 1 below. DNA binding reactions contained 170 mM NaCl, 1 mM CaCl₂and 25 mM Tris, pH 8.0. Pae3192 was incubated separately with each ofthe three duplexes for 15 minutes at 40° C. before being run on a 1%agarose gel. TABLE 1 Oligonucleotides Name (composition) Sequence Oligo1a CAGACTGGAATTCAAGCGCGAGCTCGAATAAGAGCTACTGTT (DNA) Oligo 2aAACAGTAGCTCTTATTCGAGCTCGCGCTTGAATTCCAGTCTG (DNA) Oligo 1bCAGACUGGAAUUCAAGCGCGAGCUCGAAUAAGAGCUACUGUU (RNA) Oligo 2bAACAGUAGCUCUUAUUCGAGCUCGCGCUUGAAUUCCAGUCUG (RNA) Oligo 3aGTAAAACGACGGCCAGT-3′-6FAM (DNA) Oligo 3b CUAAAACGACGGCCAGU-3′-6FAM (RNA)Oligo 4 5′-Dabsyl-ACTGGCCGTCGTTTTAC (DNA)

The results are shown in FIG. 2. FIG. 2A shows the results for theDNA:DNA duplex and the DNA:RNA duplex. FIG. 2B shows the results for thethe DNA:DNA duplex and the RNA:RNA duplex in which 20U RNasin Plus(Promega) RNase inhibitor was also included in the binding reaction.Those results show that Pae3192 gel-shifted both the DNA:DNA duplex andthe DNA:RNA duplex, but did not gel-shift the RNA:RNA duplex.

2. Tm Experiments

The ability of Pae3192 to stabilize a DNA:DNA duplex and a DNA:RNAduplex at elevated temperatures was tested. The DNA oligonucleotide 3a,RNA nucleotide 3b, and DNA oligonucleotide 4 of Table 1 above were usedin this experiment. Oligonucleotides 3a and 3b included a fluorophore(FAM) and oligonucleotide 4 included a quencher (Dabsyl). Annealing ofoligonucleotide 4 to either oligonucleotide 3a or oligonucleotide 3bresults in quenching of the fluorophore, because the oligonucleotidesare brought into close proximity. Melting can thus be monitored in areal-time PCR apparatus as in increase in fluorescence. Tm's wereassigned as the minima of the negative derivative of the fluorescenceversus temperature curves.

Pae3192 was separately incubated with the DNA:DNA duplex or with theDNA:RNA duplex for 20 minutes at 20° C. in the presence of a proteinbuffer containing 15 mM NaCl, 88 uM CaCI₂ and 50 mM Tris, pH 8.0.Pae3192 was present at 12.5 uM (88 ug/ml), while the duplexes were at0.25 uM. A dissociation curve (25° C. to 95° C.) was then taken usingthe AB 7900 apparatus. Negative controls were also monitored in whichthe protein buffer was added alone or the protein buffer plus 88 ug/mlof bovine serum albumin (BSA) was added. Overall, the addition of BSAhad no effect on the Tm's of the duplexes (not shown). The observeddifferences in Tm between the buffer only samples and thePae3192-containing samples are indicated in Table 2. Pae3192 stabilizedboth DNA:DNA duplexes and DNA:RNA hybrids, though stabilization ofDNA:RNA duplex occured to a slightly lesser extent. TABLE 2Stabilization of DNA:DNA and DNA:RNA duplexes by Pae3192. Tm's (° C.)for annealed oligos 3a + 4 (DNA:DNA) or oligos 3b + 4 (DNA:RNA) in thepresence or absence of Pae3192 are indicated. T_(m), buffer aloneT_(m), + Pae3192 ΔT_(m) DNA:DNA 57.5 75.9 18.4 DNA:RNA 56.8 71.1 14.3

Sso7d has also been shown to have DNA:DNA duplex stabilization activity(see, for example, McAfee et al, Biochemistry, 34(31):10063-10077(1995).

Together with the data below in Example L that showed that thePae3192-Pfu fusion protein possessed an acquired reverse transcriptase(RT) activity, these data in Example K(1) and (2) support the conclusionthat Pae3192 binds to RNA:DNA duplexes.

L. Use of 10His-Pfu-Pae3192 and 10His-Pfu-Pae3192, Exo-Minus Version inRT-PCR

RT-PCR reactions were performed. All reagents, including RNA template,primers, dNTPs and buffers, were from the GeneAmp EZ rTth RT-PCR Kit(P/N N808-0179; Applied Biosystems, Foster City, Calif.). The enzymesthat were tested were Taq DNA polymerase (AmpliTaq; Applied Biosystems,Inc); rtth DNA polymerase (included with the GeneAmp EZ rTth RT-PCRKit); Phusion DNA polymerase (Finnzymes); 10His-Pfu-Pae3192 (describedin Example C(1)(a) above); 10His-Pfu-Pae3192, exo-minus version(described in Example C(1)(d) above (a double mutant of10His-Pfu-Pae3192 rendering the activity of the 3′→5′ exonuclease domainessentially inactive)), and P.fu polymerase (without nucleic acidbinding polypeptide) (Stratagene).

Each of the enzymes was used in reactions employing the standard RT-PCRcycling conditions recommended by the manufacturer. AmpliTaq, rtth,10His-Pfu-Pae3192, and 10His-Pfu-Pae3192, exo-minus version, eachprovided PCR amplification product from the starting RNA template (datanot shown). Pfu polymerase (without nucleic acid binding polypeptide)did not amplify a product (data not shown).

A RT-PCR reaction was also performed with each of the enzymes accordingto the manufacturer's instructions, with the following modifications tothe cycling parameters: the initial RT step was shortened from 30minutes to 5 minutes; the two step PCR cycling program was shortened sothat the holding time at both temperatures was reduced to 2 secondseach; and the final extension at 72° C. was omitted. As shown in FIG. 3,when the RT-PCR cycling conditions were significantly shortened asdescribed above, only 10His-Pfu-Pae3192 and 10His-Pfu-Pae3192, exo-minusversion, yielded a significant amount of amplification product (lanes 6,7, 8 in FIG. 3); the rtth enzyme (lane 4) no longer produced a band andAmpliTaq (lane 3) produced a greatly reduced yield.

M. Use of 10His-Pae3192-Taq in PCR

Three sets of PCR reactions were performed. All reaction mixturescontained lambda DNA as the template and the following forward andreverse primers: (SEQ ID NO:47) 5′-AGCCAAGGCCAATATCTAAGTAAC-3′ (Tm=54.1°C.) (SEQ ID NO:48) 5′-CGAAGCATTGGCCGTAAGTG-3′ (Tm=58.4° C.)

The first set of reaction mixtures was prepared as follows: Component(stock concentration) Volume Final concentration Lambda (λ) DNA 1 μl 0.2ng/μl (10 ng/μl) dNTPs (2.5 mM each) 1 μl 200 μM each Buffer* (10×) 5 μl1× Forward primer 1 μl 0.2 μM (10 μM) Reverse primer 1 μl 0.2 μM (10 μM)Enzyme 0.5 μl dH₂O 40.5 μl 50 μl final volume1× Buffer*: 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂

In separate reaction mixtures, the enzymes AmpliTaq (Roche MolecularSystems, Pleasanton, Calif.) and 10His-Pae3192-Taq (described in ExampleC(2)(a) above) were tested. Two-fold serial dilutions of the10His-Pae3192-Taq were tested in the range of 24, 12, 6, 3, and 1.5Units per 50 uL reaction. AmpliTaq was tested at 2.5 Units per 50 uLreaction.

The first set of reaction mixtures were subjected to PCR cyclingconditions using an Applied Biosystems 9700 Thermal Cycler (AppliedBiosystems, Foster City, Calif.), as follows: 95° C., 1 min; 94° C., 30sec; 55° C., 30 sec; and {close oversize bracket} 30 cycles 72° C., 1sec. 72° C., 10 min

After the 30 cycles, the reaction mixtures were analyzed by agarose gelelectrophoresis. AmpliTaq provided PCR amplification product from thestarting template (data not shown). The 10His-Pae3192-Taq did notamplify a product (data not shown).

The second set of reaction mixtures was identical to the first set ofreaction mixtures discussed above except that the 1× Buffer* contained15 mM Tris-HCl pH 8.9, 90 mM KCl, 1.5 mM MgCl₂, and 0.05% Tween 20.

The enzyme 10His-Pae3192-Taq (described in Example C(2)(a) above) wastested. Two-fold serial dilutions of the 10His-Pae3192-Taq were testedin the range of 24, 12, 6, 3, and 1.5 Units per 50 uL reaction.

The second set of reaction mixtures were subjected to same PCR cyclingconditions discussed above for the first set of reaction mixtures usingan Applied Biosystems 9700 Thermal Cycler (Applied Biosystems, FosterCity, Calif.).

After the 30 cycles, the reaction mixtures were analyzed by agarose gelelectrophoresis. See FIG. 4. The 10His-Pae3192-Taq amplified a productas shown in FIG. 4.

The third set of reaction mixtures was prepared as follows: Component(stock concentration) Volume Final concentration Lambda (λ) DNA 1 μl 0.2ng/μl (10 ng/μl) dNTPs (2.5 mM 1 μl 200 μM each each) Buffer* (5×) 10 μl1× Forward primer 1 μl 0.2 μM (10 μM) Reverse primer 1 μl 0.2 μM (10 μM)Enzyme 0.5 μl dH₂O 36.5 μl 50 μl final volume

1× Buffer* for 10His-Pae3192-Taq: 15 mM Tris-HCl at indicated pH, 90 mMKCl, 1.5 mM MgCl₂, and some reactions further included 0.05% Tween 20 inthe buffer, while others included no Tween 20 in the buffer (pH valuesof 7.55; 7.7; 8.2; 8.6; 8.7; 9.07; and 9.3 were tested)

1× Buffer* for AmpiTaq: 10 mM Tris-HCl at indicated pH, 50 mM KCl, 1.5mM MgCl₂ (pH values of 7.55; 7.7; 8.2; 8.6; 8.7; 9.07; and 9.3 weretested)

In separate reaction mixtures, the enzymes AmpliTaq (Roche MolecularSystems, Pleasanton, Calif.) and 10His-Pae3192-Taq (described in ExampleC(2)(a) above) were tested. The 10His-Pae3192-Taq was tested at 2.5Units per 50 uL reaction. AmpliTaq was tested at 2.5 Units per 50 uLreaction.

The third set of reaction mixtures were subjected to same PCR cyclingconditions discussed above for the first set of reaction mixtures usingan Applied Biosystems 9700 Thermal Cycler (Applied Biosystems, FosterCity, Calif.).

After the 30 cycles, the reaction mixtures were analyzed by agarose gelelectrophoresis. As shown in FIG. 5, AmpliTaq provided PCR amplificationproduct at the lower pH levels tested, but did not provide PCRamplification product at the higher pH levels tested. As shown in FIG.5, 10His-Pae3192-Taq with Tween 20 in the buffer provided PCRamplification product at the higher pH levels tested. The10His-Pae3192-Taq without Tween 20 in the buffer did not provide PCRamplification product

The 0.05% Tween can also be substituted with 0.05% NP-40 with similaractivity in PCR (data not shown). SEQ ID NO: Brief Description Sequence1 Pae3192 MSKKQKLKFYDIKAKQAFETDQYEVIEKQTARGPMMFAVAKSPYTGIKVYRLLGKKK(protein) 2 PAE3192 atgtccaaga agcagaaact aaagttctac gacataaaggcgaagcaggc (ORF) gtttgagact gaccagtacg aggttattga gaagcagact gcccgcggtccgatgatgtt cgccgtggcc aaatcgccgt acaccggcat aaaagtatac agactgttaggcaagaagaa ataa 3 PAE3289 atgtccaaga agcagaaact aaagttctac gacataaaggcgaagcaggc (ORF) gtttgagact gaccagtacg aggttattga gaagcagact gcccgcggtccgatgatgtt cgccgtggcc aaatcgccgt acaccggcat aaaagtatac agactattaggcaagaagaa ataa 4 Pae0384MAKQKLKFYDIKAKQSFETDKYEVIEKETARGPMLFAVATSPYTGIKVYRLLGKKK (protein) 5PAE0384 atggccaaac aaaaactaaa gttctacgac ataaaagcga aacagtcctt (ORF)cgaaacggac aaatacgagg tcattgagaa agagacggcc cgcgggccga tgttatttgcagtggcaacc tcgccgtaca ctggcataaa ggtgtacaga ctgttaggca agaagaaata a 6Ape3192 MPKKEKIKFFDLVAKKYYETDNYEVEIKETKRGKFRFAKAKSPYTGIKIFYRVLGKA 7APE3192 atgcccaaga aggagaagat aaagttcttc gacctagtcg ccaagaagta (ORF)ctacgagact gacaactacg aagtcgagat aaaggagact aagaggggca agtttaggttcgccaaagcc aagagcccgt acaccggcaa gatcttctat agagtgctag gcaaagccta g 8p3192-a atgtccaaga agcagaaact gaagttctac gacattaagg cgaagcaggc gtttgag 9p3192-b accgaccagt acgaggttat tgagaagcag accgcccgcg gtccgatgat gttcgcc10 p3192-c gtggccaaat cgccgtacac cggcattaaa gtgtaccgcc tgttaggcaagaagaaataa 11 p3192-y gtactggtcg gtctcaaacg cctg 12 p3192-z cgatttggccacggcgaaca tcat 13 8, 9, and 10 atgtccaaga agcagaaact gaagttctacgacattaagg cgaagcaggc assembled gtttgagacc gaccagtacg aggttattgagaagcagacc gcccgcggtc cgatgatgtt cgccgtggcc aaatcgccgt acaccggcattaaagtgtac cgcctgttag gcaagaagaa ataa 14 ap3192-a atgccgaaga aggagaagattaagttcttc gacctggtcg ccaagaagta ctacgag 15 ap3192-b actgacaactacgaagtcga gattaaggag actaagcgcg gcaagtttcg cttcgcc 16 ap3192-caaagccaaga gcccgtacac cggcaagatc ttctatcgcg tgctgggcaa agcctag 17ap3192-y gtagttgtca gtctcgtagt actt 18 ap3192-z gctcttggct ttggcgaagcgaaa 19 14,15, and atgccgaaga aggagaagat taagttdttc gacctggtcgccaagaagta 16 assembled ctacgagact gacaactacg aagtcgagat taaggagactaagcgcggca agtttcgctt cgccaaagcc aagagcccgt acaccggcaa gatcttctatcgcgtgctgg gcaaagccta g 20 Sso7dMATVKFKYKGEEKQVDISKIKKVWRVGKMTSFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKK 21Sso7d METSMATVKFKYKGEEKQVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEvariant KQKK 22 polynucleotideccatgggccatcatcatcatcatcatcatcatcatcacagcagcggccatatcgaaggtc encoding10His- gtcatatgattttagatgtggattacataactgaagaaggaaaacctgttatt~ggctatPfu-Pae3192 tcaaaaaagagaacggaaaatttaagatagagcatgatagaacttttagaccatacatttacgctcttctcagggatgattcaaagattgaagaagttaagaaaataacgggggaaaggcatggaaagattgtgagaattgttgatgtagagaaggttgagaaaaagtttctcggcaagcctattaccgtgtggaaactttatttggaacatccccaagatgttcccactattagagaaaaagttagagaacatccagcagttgtggacatcttcgaatacgatattccatttgcaaagagatacctcatcgacaaaggcctaataccaatggagggggaagaagagctaaagattcttgccttcgatatagaaaccctctatcacgaaggagaagagtttggaaaaggcccaattataatgattagttatgcagatgaaaatgaagcaaaggtgattacttggaaaaacatagatcttccatacgttgaggttgtatcaagcgagagagagatgataaagagatttctcaggattatcagggagaaggatcctgacattatagttacttataatggagactcattcgacttcccatatttagcgaaaagggcagaaaaacttgggattaaattaaccattggaagagatggaagcgagcccaagatgcagagaataggcgatatgacggctgtagaagtcaagggaagaatacatttcgacttgtatcatgtaataacaaggacaataaatctcccaacatacacactagaggctgtatatgaagcaatttttggaaagccaaaggagaaggtatacgccgacgagatagcaaaagcctgggaaagtggagagaaccttgagagagttgccaaatactcgatggaagatgcaaaggcaacttatgaactcgggaaagaattccttccaatggaaattcagctttcaagattagttggacaacctttatgggatgtttcaaggtcaagcacagggaaccttgtagagtggttcttacttaggaaagcctacgaaagaaacgaagtagctccaaacaagccaagtgaagaggagtatcaaagaaggctcagggagagctacacaggtggattcgttaaagagccagaaaaggggttgtgggaaaacatagtatacctagattttagagccctatatccctcgattataattacccacaatgtttctcccgatactctaaatcttgagggatgcaagaactatgatatcgctcctcaagtaggccacaagttctgcaaggacatccctggttttataccaagtctcttgggacatttgttagaggaaagacaaaagattaagacaaaaatgaaggaaactcaagatcctatagaaaaaatactccttgactatagacaaaaagcgataaaactcttagcaaattctttctacggatattatggctatgcaaaagcaagatggtactgtaaggagtgtgctgagagcgttactgcctggggaagaaagtacatcgagttagtatggaaggagctcgaagaaaagtttggatttaaagtcctctacattgacactgatggtctctatgcaactatcccaggaggagaaagtgaggaaataaagaaaaaggctctagaatttgtaaaatacataaattcaaagctccctggactgctagagcttgaatatgaagggttttataagaggggattcttcgttacgaagaagaggtatgcagtaatagatgaagaaggaaaagtcattactcgtggtttagagatagttaggagagattggagtgaaattgcaaaagaaactcaagctagagttttggagacaatactaaaacacggagatgttgaagaagctgtgagaatagtaaaagaagtaatacaaaagcttgccaattatgaaattccaccagagaagctcgcaatatatgagcagataacaagaccattacatgagtataaggcgataggtcctcacgtagctgttgcaaagaaactagctgctaaaggagttaaaataaagccaggaatggtaattggatacatagtacttagaggcgatggtccaattagcaatagggcaattctagctgaggaatacgatcccaaaaagcacaagtatgacgcagaatattacattgagaaccaggttcttccagcggtacttaggatattggagggatttggatacagaaaggaagacctcagataccaaaagacaagacaagtcggcctaacttcctggcttaacattaaaaaatccggtaccggcggtggcggtatgtccaagaagcagaaactgaagttctacgacattaaggcgaagcaggcgtttgagaccgaccagtacgaggttattgagaagcagaccgcccgcggtccgatgatgttcgccgtggccaaatcgccgtacaccggcattaaagtgtaccgcctgttaggcaagaagaaat aactcgag 23amino acid MGHHHHHHHHHHSSGHIEGRHMILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYsequence of ALLRDDSKIEEVKKITGERHGKIVRTVDVEKVEKKFLGKPTTVWKLYLEHPQDvPTIREK10His-Pfu- VREHPAVVDTFEYDIPFAKRYLTDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPITMPae3192 ISYADENEAKVTTWKNTDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGTKLTTGRDGSEPKMQRIGDMTAVEVKGRTHFDLYHVITRTINLPTYTLEAVYEATFGKPKEKVYADETAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENTVYLDFRALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGPIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKATGPRVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKSGTGGGGMSKKQKLKFYDIKAKQAFETDQYEVTEKQTARGPMMKAVAKSPYTGIKVYRLLGKKK 24 amino acidHMTLDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIY sequence ofALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREK Pfu-Pae3192VREHPAVVDTFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDTETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTTGRDGSEPKMQRTGDMTAVEVKGRIHFDLYHVITRTTNLPTYTLEAVYEATFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSITITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFTPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYTDTDGLYATIPGGESEETKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLETVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVTQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYTENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKSGTGGGGMSKKQKLKFYDIKAKQAFETDQYEVTEKQTARGPMMFAVAKSPYTGIKVYRLLGKKK 25 polynucleotideccatgggccatcatcatcatcatcatcatcatcatcacagcagcggccatatcgaaggtc encoding10His- gtcatatgattttagatgtggattacataactgaagaaggaaaacctgttattaggctatPfu-Ape3192 tcaaaaaagagaacggaaaatttaagatagagcatgatagaacttttagaccatacatttacgctcttctcagggatgattcaaagattgaagaagttaagaaaataacgggggaaaggcatggaaagattgtgagaattgttgatgtagagaaggttgagaaaaagtttctcggcaagcctattaccgtgtggaaactttatttggaacatccccaagatgttcccactattagagaaaaagttagagaacatccagcagttgtggacatcttcgaatacgatattccatttgcaaagagatacctcatcgacaaaggcctaataccaatggagggggaagaagagctaaagattcttgccttcgatatagaaaccctctatcacgaaggagaagagtttggaaaaggcccaattataatgattagttatgcagatgaaaatgaagcaaaggtgattacttggaaaaacatagatcttccatacgttgaggttgtatcaagcgagagagagatgataaagagatttctcaggattatcagggagaaggatcctgacattatagttacttataatggagactcattcgacttcccatatttagcgaaaagggcagaaaaacttgggattaaattaaccattggaagagatggaagcgagcccaagatgcagagaataggcgatatgacggctgtagaagtcaagggaagaatacatttcgacttgtatcatgtaataacaaggacaataaatctcccaacatacacactagaggctgtatatgaagcaatttttggaaagccaaaggagaaggtatacgccgacgagatagcaaaagcctgggaaagtggagagaaccttgagagagttgccaaatactcgatggaagatgcaaaggcaacttatgaactcgggaaagaattccttccaatggaaattcagctttcaagattagttggacaacctttatgggatgtttcaaggtcaagcacagggaaccttgtagagtggttcttacttaggaaagcctacgaaagaaacgaagtagctccaaacaagccaagtgaagaggagtatcaaagaaggctcagggagagctacacaggtggattcgttaaagagccagaaaaggggttgtgggaaaacatagtatacctagattttagagccctatatccctcgattataattacccacaatgtttctcccgatactctaaatcttgagggatgcaagaactatgatatcgctcctcaagtaggccacaagttctgcaaggacatccctggttttataccaagtctcttgggacatttgttagaggaaagacaaaagattaagacaaaaatgaaggaaactcaagatcctatagaaaaaatactccttgactatagacaaaaagcgataaaactcttagcaaattctttctacggatattatggctatgcaaaagcaagatggtactgtaaggagtgtgctgagagcgttactgcctggggaagaaagtacatcgagttagtatggaaggagctcgaagaaaagtttggatttaaagtcctctacattgacactgatggtctctatgcaactatcccaggaggagaaagtgaggaaataaagaaaaaggctctagaatttgtaaaatacataaattcaaagctccctggactgctagagcttgaatatgaagggttttataagaggggattcttcgttacgaagaagaggtatgcagtaatagatgaagaaggaaaagtcattactcgtggtttagagatagttaggagagattggagtgaaattgcaaaagaaactcaagctagagttttggagacaatactaaaacacggagatgttgaagaagctgtgagaatagtaaaagaagtaatacaaaagcttgccaattatgaaattccaccagagaagctcgcaatatatgagcagataacaagaccattacatgagtataaggcgataggtcctcacgtagctgttgcaaagaaactagctgctaaaggagttaaaataaagccaggaatggtaattggatacatagtacttagaggcgatggtccaattagcaatagggcaattctagctgaggaatacgatcccaaaaagcacaagtatgacgcagaatattacattgagaaccaggttcttccagcggtacttaggatattggagggatttggatacagaaaggaagacctcagataccaaaagacaagacaagtcggcctaacttcctggcttaacattaaaaaatccggtaccggcggtggcggtccgaagaaggagaagattaggttcttcgacctggtcgccaagaagtactacgagactgacaactacgaagtcgagattaaggagactaagcgcggcaagtttcgcttcgccaaagccaagagcccgtacaccggcaagatcttctatcgcgtgctgggcaaagcctaactcgag 26 aminoacid MGHHHHHHHHHHSSGHTEGRHMTLDVDYITEEGKPVIRLFKKENGKFKTEHDRTFRPYIYsequence of ALLRDDSKIEEVKKTTGERHGKTVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREK10His-Pfu- VREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMApe3192 ISYADENEAKVITWKNTDLPYVEVVSSEREMTKRFLRTTREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTTGRDGSEPKMQRTGDMTAVEVKGRIHFDLYHVTTRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEETPMEIQLSRLVGQPLWDVSRSSTGNLUEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENTVYLDFRALYPSTIITHNVSPDTLNLEGCKNYDTAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPTEKILLDYRQKATKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYTDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVTDEEGKVITRGLETVRRDWSETAKETQARVLETILKHGDVEEAVRTVKEVTQKLANYETPPEKLATYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVTGYTVLRGDGPTSNRAILAEEYDPKKHKYDAEYYTENQVLPAVLRTLEGFGYRKEDLRYQKTRQVGLTSWLNTKKSGTGGGGPKKEKIRFFDLVAKKYYETDNYEVEIKETKRGKFRFAKAKSPYTGKTFYRVLGKA 27 amino acid HMTLDVDYTTEEGKPVTRLFKKENGKFKIEHDRTFRPYIYsequence of ALLRDDSKTEEVKKITGERHGKIVRIVDVEKVEKKFLGKPTTVWKLYLEHPQDVPTIREKPfu-Ape3192 VREHPAVVDTFEYDIPFAKRYLIDKGLIPMEGEEELKTLAFDTETLYHEGEEFGKGPTIMTSYADENEAKVTTWKNTDLPYVEVVSSEREMTKRFLRITREKDPDITVTYNGDSFDFPYLAKRAEKLGTKLTIGRDGSEPKMQRTGDMTAVEVKGRIHFDLYHVTTRTINLPTYTLEAVYEATFGKPKEKVYADETAKAWESGENLERVAKYSMEDAKATYELGKEFLPMETQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENTVYLDFRALYPSTTTTHNVSPDTLNLEGCKNYDTAPQVGHKFCKDTPGFTPSLLGHLLEERQKTKTKMKETQDPTEKTLLDYRQKATKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYTELVWKELEEKFGFKVLYTDTDGLYATTPGGESEETKKKALEFVKYTNSKLPGLLELEYEGFYKRGFFVTKKRYAVTDEEGKVTTRGLETVRRDWSETAKETQARVLETTLKHGDVEEAVRTVKEVTQKLANYETPPEKLATYEQTTRPLHEYKATGPHVAVAKKLAAKGVKIKPGMVTGYTVLRGDGPTSNRATLAEEYDPKKHKYDAEYYTENQVLPAVLRTLEGFGYRKEDLRYQKTRQVGLTSWLNTKKSGTGGGGPKKEKTRFFDLVAKKYYETDNYEVETKETKRGKFRFAKAKSPYTGKIFYRVLGKA 28 Pae/ApeKXKXKFXDXXAKXXXETDXYEVXXKXTXRGXXXFAXAKSPYTGXXXYRXLGK consensus sequence29 oligo for gttttcccagtcacgacgttgtaaaacgacggcc processivity assay 30Pfu DNA MTLDVDYTTEEGKPVIRLFKKENGKFKIEHDRTFRPYIY polymeraseALLRDDSKTEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGTKLTTGRDGSEPKMQRTGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMETQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLwENIVYLDERALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDTPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGPYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSETAKETQARVLETILKHGDVEEAVRTVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVTGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKS 31 Taq DNAMRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDG polymeraseDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDTHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGTGED WLSAKE 32polynucleotideATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCG encoding10His- TCATATGTCCAAGAAGCAGAAACTGAAGTTCTACGACATTAAGGCGAAGCAGGCGTTTGPae3192-Taq AGACCGACCAGTACGAGGTTATTGAGAAGCAGACCGCCCGCGGTCCGATGATGTTCGCCGTGGCCAAATCGCCGTACACCGGCATTAAAGTGTACCGCCTGTTAGGCAAGAAGAAAGGCGGCGGTGTCACTAGTGGGATGCTGCCCCTCTTTGAGCCCAAGGGCCGGGTCCTCCTGGTGGACGGCCACCACCTGGCCTACCGCACCTTCCACGCCCTGAAGGGCCTCACCACCAGCCGGGGGGAGCCGGTGCAGGCGGTCTACGGCTTCGCCAAGAGCCTCCTCAAGGCCCTCAAGGAGGACGGGGACGCGGTGATCGTGGTCTTTGACGCCAAGGCCCCCTCCTTCCGCCACGAGGCCTACGGGGGGTACAAGGCGGGCCGGGCCCCCACGCCGGAGGACTTTCCCCGGCAACTCGCCCTCATCAAGGAGCTGGTGGACCTCCTGGGGCTGGCGCGCCTCGAGGTCCCGGGCTACGAGGCGGACGACGTCCTGGCCAGCCTGGCCAAGAAGGCGGAAAAGGAGGGCTACGAGGTCCGCATCCTCACCGCCGACAAAGACCTTTACCAGCTCCTTTCCGACCGCATCCACGTCCTCCACCCCGAGGGGTACCTCATCACCCCGGCCTGGCTTTGGGAAAAGTACGGCCTGAGGCCCGACCAGTGGGCCGACTACCGGGCCCTGACCGGGGACGAGTCCGACAACCTTCCCGGGGTCAAGGGCATCGGGGAGAAGACGGCGAGGAAGCTTCTGGAGGAGTGGGGGAGCCTGGAAGCCCTCCTCAAGAACCTGGACCGGCTGAAGCCCGCCATCCGGGAGAAGATCCTGGCCCACATGGACGATCTGAAGCTCTCCTGGGACCTGGCCAAGGTGCGCACCGACCTGCCCCTGGAGGTGGACTTCGCCAAAAGGCGGGAGCCCGACCGGGAGAGGCTTAGGGCCTTTCTGGAGAGGCTTGAGTTTGGCAGCCTCCTCCACGAGTTCGGCGTTCTGGAAAGCCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCGGCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGCCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGGTCCTCTACGGCATGTCGCCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGACTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGTGA 33 amino acidMGHHHHHHHHHHSSGHIEGRHMSKKQKLKFYDTKAKQAFETDQYEVIEKQTARGPMMFA sequence ofVAKSPYTGIKVYRLLGKKKGGGVTSGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTS10His-Pae3192-RGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQ TaqLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLTTPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQTELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTTNFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLANVKLEPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE 34 amino acidHMSKKQKLKEYDIKAKQAFETDQYEVTEKQTARGPMMFA sequence ofVAKSPYTGTKVYRLLGKKKGGGVTSGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTS Pae3192-TaqRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGTGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKTLQYRELTKLKSTYIDPLPDLTHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRTRPAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERNAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE 35 polynucleotideATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAG encoding 10-GTCGTCATATGCCGAAGAAGGAGAAGATTAAGTTCTTCGACCTGGTCGCCAAGAAGTAC His-Ape3192-TACGAGACTGACAACTACGAAGTCGAGATTAAGGAGACTAAGCGCGGCAAGTTTCGCTT TaqCGCCAAAGCCAAGAGCCCGTACACCGGCAAGATCTTCTATCGCGTGCTGGGCAAAGCCGGCGGCGGTGTCACTAGTGGGATGCTGCCCCTCTTTGAGCCCAAGGGCCGGGTCCTCCTGGTGGACGGCCACCACCTGGCCTACCGCACCTTCCACGCCCTGAAGGGCCTCACCACCAGCCGGGGGGAGCCGGTGCAGGCGGTCTACGGCTTCGCCAAGAGCCTCCTCAAGGCCCTCAAGGAGGACGGGGACGCGGTGATCGTGGTCTTTGACGCCAAGGCCCCCTCCTTCCGCCACGAGGCCTACGGGGGGTACAAGGCGGGCCGGGCCCCCACGCCGGAGGACTTTCCCCGGCAACTCGCCCTCATCAAGGAGCTGGTGGACCTCCTGGGGCTGGCGCGCCTCGAGGTCCCGGGCTACGAGGCGGACGACGTCCTGGCCAGCCTGGCCAAGAAGGCGGAAAAGGAGGGCTACGAGGTCCGCATCCTCACCGCCGACAAAGACCTTTACCAGCTCCTTTCCGACCGCATCCACGTCCTCCACCCCGAGGGGTACCTCATCACCCCGGCCTGGCTTTGGGAAAAGTACGGCCTGAGGCCCGACCAGTGGGCCGACTACCGGGCCCTGACCGGGGACGAGTCCGACAACCTTCCCGGGGTCAAGGGCATCGGGGAGAAGACGGCGAGGAAGCTTCTGGAGGAGTGGGGGAGCCTGGAAGCCCTCCTCAAGAACCTGGACCGGCTGAAGCCCGCCATCCGGGAGAAGATCCTGGCCCACATGGACGATCTGAAGCTCTCCTGGGACCTGGCCAAGGTGCGCACCGACCTGCCCCTGGAGGTGGACTTCGCCAAAAGGCGGGAGCCCGACCGGGAGAGGCTTAGGGCCTTTCTGGAGAGGCTTGAGTTTGGCAGCCTCCTCCACGAGTTCGGCCTTCTGGAAAGCCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCCCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAAAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGGTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGTGA 36 amino acidMGHHHHHHHHHHSSGHIEGRHMPKKEKIKFFDLVAKKYYETDNYEVEIKETKRGKERFA sequence of10- KAKSPYTGKTFYRVLGKAGGGVTSGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRHis-Ape3192- GEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLTaq ALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRTLTADKDLYQLLSDRTHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGTGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKTLQYRELTKLKSTYTDPLPDLIHPRTGRLhTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDTHTETASWMFGVPREAVDPLMRRAAKTTNFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGTGEDWLSAKE 37 amino acidHMPKKEKIKFFDLVAKKYYETDNYEVETKETKRGKFRFA sequence ofKAKSPYTGKIFYRVLGKAGGGVTSGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSR Ape3192-TaqGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDEPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKTLQYRELTKLKSTYTDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNTPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLTRVFQEGRDTHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELATPYEEAQAFIERYEQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGTGEDWLSAKE 38 polynucleotideATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCG encoding10His- TCATATGTCCAAGAAGCAGAAACTGAAGTTCTACGACATTAAGGCGAAGCAGGCGTTTGPae3192-Taq_(ST)AGACCGACCAGTACGAGGTTATTGAGAAGCAGACCGCCCGCGGTCCGATGATGTTCGCCGTGGCCAAATCGCCGTACACCGGCATTAAAGTGTACCGCCTGTTAGGCAAGAAGAAAGGCGGCGGTGTCACTAGTCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGGCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGGTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTGGGCGTCGCGCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGGTCCTCTACGGCATGTCGGCCCACCGCCTGTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGGCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTGTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCAGCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGTGA 39 amino acidMGHHHHHHHHHHSSGHTEGRHMSKKQKLKFYDTKAKQAFETDQYEVTEKQTARGPMMFA sequence ofVAKSPYTGIKVYRLLGKKKGGGVTSPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLAL10His-Pae3092-AAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPS Taq_(ST)NTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYTDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRTRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE 40 amino acidHMSKKQKLKFYDIKAKQAFETDQYEVTEKQTARGPMMFA sequence ofVAKSPYTGIKVYRLLGKKKGGGVTSPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALPae3192-Taq_(ST)AAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPTVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRTRRAFIAEEGWLLVALDYSQTELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELATPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVG IGEDWLSAKE 41 polynucleotideATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCG encoding10His- TCATATGCCGAAGAAGGAGAAGATTAAGTTCTTCGACCTGGTCGCCAAGAAGTACTACGApe3192-Taq_(ST)AGACTGACAACTACGAAGTCGAGATTAAGGAGACTAAGCGCGGCAAGTTTCGCTTCGCCAAAGCCAAGAGCCCGTACACCGGCAAGATCTTCTATCGCGTGCTGGGCAAAGCCGGCGGCGGTGTCACTAGTCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACGCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGGCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAAGCTGATCCGGGTCTTGCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTGGCAGGAGCTAGGCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCGGTCCAGGGCACCGCCGGCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGGGAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGTGA 42 amino acidMGHHHHHHHHHHSSGHIEGRHMPKKEKIKFFDLVAKKYYETDNYEVETKETKRGKFRFA sequence ofKAKSPYTGKIFYRVLGKAGGGVTSPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALA10His-Ape3192-AARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSN Taq_(ST)TTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLTHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLTRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELATPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE 43 amino acidHMPKKEKTKFFDLVAKKYYETDNYEVEIKETKRGKFRFA sequence ofKAKSPYTGKIFYRVLGKAGGGVTSPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAApe3192-Taq_(ST)AARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPATGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELATPYEEAQAFTERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE 44 polynucleotide atggcaacagtaaagttc aagtacaaag gagaagagaag (1 of 2) caagtagata taagtaagataaagaaggta tggagagtag gcaaaatgat encoding Sso7d aagcttcacc tatgatgagggtggaggaaa gactggtaga ggagctgtaa (SEQ ID NO:20) gcgagaaaga cgctccaaaagaactactac aaatgttaga gaagcaaaag aagtaa 45 polynucleotide atggcaacagtaaagttc aagtataaag gagaagaaaaa (2 of 2) caagtagaca taagtaagataaagaaggta tggagagtcg gaaagatgat encoding Sso7d aagctttacc tatgatgagggtggaggaaa gactggtaga ggagcagtaa (SEQ ID NO:20) gcgagaaaga tgctccaaaagagctattac aaatgttaga gaaacaaaag aagtaa 46 polynucleotide ttggagatatcaatggcaac agtaaagttc aagtacaagg gagaagagaag encoding Sso7d gaagtagatataagtaagat aaagaaggta tggagagtag gcaaaatgat variant aagtttcacctatgatgagg gtggaggaaa gactggtaga ggagctgtaa (SEQ ID NO:21) gcgagaaagacgctccaaaa gaactactac aaatgttaga aaagcaaaag aaataa 47 forward primerAGCCAAGGCCAATATCTAAGTAAC 48 reverse primer CGAAGCATTGGCCGTAAGTG 49 aminoacid MGHHHHHHHHHHSSGHIEGRHMILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYTYsequence of ALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTTREK10His-Pfu- VREHPAVVDTFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPTIMSso7d ISYADENEAKVITWKNIDLPYVEVVSSEREMTKRFLRIIREKDPDITVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPKMQRTGDMTAVEVKGRIHFDLYHVTTRTINLPTYTLEAVYEATFGKPKEKVYADETAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENTVYLDFRALYPSTIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPOFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKATKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYTDTDGLYATIPGGESEETKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSETAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLATYEQTTRPLHEYKATGPHVAVAKKLAAKGVKIKPGMVIGYTVLRGDGPTSNRATLAEEYDPKKHKYDAEYYTENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNTKKSGTGGGGATVKFKYKGEEKEVDISKTKKVWRVGKMTSFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKK 50 amino acidHMILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIY sequence ofALLRDDSKTEEVKKITGERHGKIVRIVDVEKVEKKFLGKPTTVWKLYLEHPQDVPTTREK Pfu-Sso7dVREHPAVVDTFEYDIPFAKRYLIDKGLIPMEGEEELKTLAFDTETLYHEGEEFGKGPTIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDTTVTYNGDSFDFPYLAKRAEKLGTKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENTVYLDFRALYPSITITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYTELVWKELEEKFGFKVLYTDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLETVRRDWSETAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLATYEQTTRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYTVLRGDGPTSNRATLAEEYDPKKHKYDAEYYTENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKSGTGGGGATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKK 51 polynucleotideCCATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCG encoding10His- TCATATGATTTTAGATGTGGATTACATAACTGAAGAAGGAAAACCTGTTATTAGGCTATTCPfu-Sso7d AAAAAAGAGAACGGAAAATTTAAGATAGAGCATGATAGAACTTTTAGACCATACATTTACGCTCTTCTCAGGGATGATTCAAAGATTGAAGAAGTTAAGAAAATAACGGGGGAAAGGCATGGAAAGATTGTGAGAATTGTTGATGTAGAGAAGGTTGAGAAAAAGTTTCTCGGCAAGCCTATTACCGTGTGGAAACTTTATTTGGAACATCCCCAAGATGTTCCCACTATTAGAGAAAAAGTTAGAGAACATCCAGCAGTTGTGGACATCTTCGAATACGATATTCCATTTGCAAAGAGATACCTCATCGACAAAGGCCTAATACCAATGGAGGGGGAAGAAGAGCTAAAGATTCTTGCCTTCGATATAGAAACCCTCTATCACGAAGGAGAAGAGTTTGGAAAAGGCCCAATTATAATGATTAGTTATGCAGATGAAAATGAAGCAAAGGTGATTACTTGGAAAAACATAGATCTTCCATACGTTGAGGTTGTATCAAGCGAGAGAGAGATGATAAAGAGATTTCTCAGGATTATCAGGGAGAAGGATCCTGACATTATAGTTACTTATAATGGAGACTCATTCGACTTCCCATATTTAGCGAAAAGGGCAGAAAAACTTGGGATTAAATTAACCATTGGAAGAGATGGAAGCGAGCCCAAGATGCAGAGAATAGGCGATATGACGGCTGTAGAAGTCAAGGGAAGAATACATTTCGACTTGTATCATGTAATAACAAGGACAATAAATCTCCCAACATACACACTAGAGGCTGTATATGAAGCAATTTTTGGAAAGCCAAAGGAGAAGGTATACGCCGACGAGATAGCAAAAGCCTGGGAAAGTGGAGAGAACCTTGAGAGAGTTGCCAAATACTCGATGGAAGATGCAAAGGCAACTTATGAACTCGGGAAAGAATTCCTTCCAATGGAAATTCAGCTTTCAAGATTAGTTGGACAACCTTTATGGGATGTTTCAAGGTCAAGCACAGGGAACCTTGTAGAGTGGTTCTTACTTAGGAAAGCCTACGAAAGAAACGAAGTAGCTCCAAACAAGCCAAGTGAAGAGGAGTATCAAAGAAGGCTCAGGGAGAGCTACACAGGTGGATTCGTTAAAGAGCCAGAAAAGGGGTTGTGGGAAAACATAGTATACCTAGATTTTAGAGCCCTATATCCCTCGATTATAATTACCCACAATGTTTCTCGCGATACTCTAAATCTTGAGGGATGCAAGAACTATGATATCGCTCCTCAAGTAGGCCACAAGTTCTGCAAGGACATCCCTGGTTTTATACCAAGTCTCTTGGGACATTTGTTAGAGGAAAGACAAAAGATTAAGACAAAAATGAAGGAAACTCAAGATGGTATAGAAAAAATAGTGCTTGACTATAGACAAAAAGCGATAAAAGTCTTAGCAAATTCTTTCTACGGATATTATGGCTATGCAAAAGCAAGATGGTACTGTAAGGAGTGTGCTGAGAGCGTTACTGCCTGGGGAAGAAAGTACATCGAGTTAGTATGGAAGGAGCTCGAAGAAAAGTTTGGATTTAAAGTCCTCTACATTGACACTGATGGTCTCTATGCAACTATCCCAGGAGGAGAAAGTCAGGAAATAAAGAAAAAGGCTCTAGAATTTGTAAAATACATAAATTCAAAGCTCCCTGGACTGCTAGAGCTTGAATATGAAGGGTTTTATAAGAGGGGATTCTTCGTTACGAAGAAGAGGTATGCAGTAATAGATGAACAAGGAAAAGTCATTACTCGTGGTTTAGAGATAGTTAGGAGAGATTGGAGTGAAATTGCAAAAGAAACTCAAGCTAGAGTTTTGGAGACAATACTAAAACACGGAGATGTTGAAGAAGCTGTGAGAATAGTAAAAGAAGTAATACAAAAGCTTGCCAATTATGAAATTCCACCAGAGAAGCTCGCAATATATGAGCAGATAACAAGACCATTACATGAGTATAAGGCGATAGGTCCTCACGTAGCTGTTGCAAAGAAACTAGCTGCTAAAGGAGTTAAAATAAAGCCAGGAATGGTAATTGAATACATAGTACTTAGAGGCGATGGTCCAATTAGCAATAGGGCAATTCTAGCTGAGGAATACGATCCCAAAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTTCTTCCAGCGGTACTTAGGATATTGGAGGGATTTGGATACAGAAAGGAAGACCTCAGATACCAAAAGACAAGACAAGTCGGCCTAACTTCCTGGCTTAACATTAAAAAATCCGGTACCGGCGGTGGCGGTGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGCGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGTAACTCGAG 52 amino acidMLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSI sequenceof KQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRV MMLVreverse EDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLtranscriptaseTWTRLPQGFFKSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHDCLDILAEAMGTRSDLTDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRMADQAAREVATRETPGTSTLLI 53polynucleotideATGGAGCATCGGCTACATGAGACCTCAAAAGAGCCAGATGTTTCTCTAGGGTCCACATGGC encodingMMLV TGTCTGATTTTCCTCAGGCCTGGGCGGAAACCOGGGGCATGGGACTCGCAGTTCGCCAAGCreverse TCCTCTGATCATACCTCTGAAAQCAACCTCTACCCCCGTGTCCATAAAACAATACCCCATGtranscriptaseTCACAAGAAGCCAGACTGGGGATCAAGCCCCACATACAGAGACTGTTGGACCACGGAATACTCGTACCCTGCCAGTCCCCCTGGAACACGCCCCTGCTACCCGTTAAGAAACCAGGGACTAATGATTATAGGCCTGTCCAGGATCTGAGAGAAGTCAACAACCGCGTGGAAGACATCCACCCCACCGTGCCCAACCCTTACAACCTCTTQAGCGGGCTCCCACCGTCCCACCAGTGGTACACTGTGCTTGATTTAAAGGATQCCTTTTTCTGCCTGAGACTCCACCCCACCAGTCAGCCTCTCTTCGCCTTTGAGTGGAGAGATCCAGAGATGGGAATCTCAGGACAATTGACCTGGACCAGACTCCCACAGGGTTTCAAAAACAGTCCCACCCTGTTTGATGAGGCACTGCACAGAGACCTAGCAGACTTCCGGATCCAGCACCCAGACTTGATCCTGCTACAGTACGTGGATGACTTACTGCTGGCCCCCACTTCTGACCTAGACTGCCAACAACGTACTCGGGCCCTGTTACAAACCCTAGGGAACCTCGGGTATCGGGCCTCGGCCAAGAAAGCCCAAATTTGCCAGAAACACGTCAAGTATCTCGGGTATCTTCTAAAAGAGGGTCAGAGATGGCTGACTGAGGCCAGAAAAGAGACTGTGATGGGGCAGCCTACTCCGAAGACCCCTCGACAACTAAGGGAGTTCCTAGGGACGGCAGGCTTCTGTCGCCTCTGGATCCCTGGGTTTGCAGAAATGGCAGCCCCCTTGTACCCTCTCACCAAAACGGGGACTCTGTTTAATTGGGGCCCAGACCAACAAAAGGCCTATCAAGAAATCAAGCAAGCTCTTCTAACTGCCCCAGCCCTGGGGTTGCCAGATTTGACTAAGCCCTTTGAACTCTTTGTCGACGAGAAGCAGGGCTACGCCAAAGGCGTCCTAACGCAAAAGCTGGGACCTTGGCCTCGGCCGGTGGCCTACCTGTCTAAAAAGCTAGACCCAGTGGCAGCTGGCTGGCCCCCCTCCCTACGGATGGTGGCAGCCATTGCAGTTCTGACAAAAGATGCTGGCAACCTCACTATGGGACAGCCGTTGGTCATTCTCGCCCCCCATGCCGTAGAGGCACTAGTTAAGCAACCCCCTGATCGCTGGCTCTCCAATGCCCGGATGACCCATTACCAAGCCCTGCTCCTGGACACGGACCGGGTCCAGTTCGGGCCAGTAGTGGCCCTAAATCCAGCTACGCTGCTCCCTCTGCCTGAGGAGGGGCTGCAACATGACTCCCTTGACATCTTCGCTGAAGCCCACGGAACTAGATCAGATCTTACGGACCAGCCCCTCCCAGACGCCGACCACACCTGGTACACGGATGGGAGCAGCTTCCTGCAAGAAGGGCAGCGTAAGGCCGGACCAGCGGTGACCACTGAGACTGAGGTAATCTGGGCCAGGGCATTGCCAGCC

1. A method of amplifying a nucleic acid sequence, wherein the methodcomprises subjecting a reaction mixture to at least one amplificationcycle, wherein the reaction mixture comprises a double-stranded nucleicacid, at least two primers capable of annealing to complementary strandsof the double-stranded nucleic acid, and a fusion protein comprising athermostable DNA polymerase and a nucleic acid binding polypeptide, andwherein the at least one amplification cycle comprises: denaturing thedouble-stranded nucleic acid; annealing the at least two primers tocomplementary strands of the denatured double-stranded nucleic acid; andextending the at least two primers; and wherein the time to complete oneamplification cycle is 20 seconds or less.
 2. (canceled)
 3. (canceled)4. The method of claim 1, wherein the annealing occurs at an annealingtemperature that is greater than the predicted Tm of at least one of theprimers.
 5. The method of claim 4, wherein the annealing temperature isat least about 5° C. greater than the predicted Tm of at least one ofthe primers.
 6. The method of claim 4, wherein the annealing temperatureis at least about 10° C. greater than the predicted Tm of at least oneof the primers. 7-9. (canceled)
 10. The method of claim 4, wherein theextending occurs at the annealing temperature.
 11. The method of claim10, wherein the reaction mixture is held at the annealing temperaturefor 1 second or less.
 12. The method of claim 10, wherein the denaturingoccurs at a denaturing temperature that is sufficient to denature thedouble-stranded nucleic acid.
 13. The method of claim 12, wherein thedenaturing temperature is from about 85° C. to about 100° C.
 14. Themethod of claim 12, wherein the reaction mixture is held at thedenaturing temperature for 1 second or less.
 15. The method of claim 14,wherein the reaction mixture is held at the denaturing temperature for 1second or less and the annealing temperature for 1 second or less. 16.The method of claim 15, wherein the denaturing comprises bringing thereaction mixture to the denaturing temperature without holding thereaction mixture at the denaturing temperature after the denaturingtemperature is reached, and bringing the reaction mixture to theannealing temperature without holding the reaction mixture at theannealing temperature after the annealing temperature is reached. 17.The method of claim 1, wherein the nucleic acid binding polypeptidecomprises an amino acid sequence of a nucleic acid binding polypeptidefrom a thermophilic microbe.
 18. The method of claim 17, wherein thenucleic acid binding polypeptide comprises an amino acid sequence of anucleic acid binding polypeptide from Sulfolobus.
 19. The method ofclaim 17, wherein the nucleic acid binding polypeptide is a Crenarchaealnucleic acid binding polypeptide.
 20. The method of claim 1, wherein thenucleic acid binding polypeptide comprises a sequence selected from: a)SEQ ID NO:20; b) a sequence having at least 80% identity to SEQ IDNO:20; c) SEQ ID NO:6; d) a sequence having at least 80% identity to SEQID NO:6; e) SEQ ID NO:1; and f) a sequence having at least 80% identityto SEQ ID NO:1.
 21. The method of claim 1, wherein the thermostable DNApolymerase comprises an archaeal family B polymerase or a fragment orvariant of an archaeal family B polymerase having polymerase activity.22. The method of claim 21, wherein the thermostable DNA polymerasecomprises Pfu polymerase or a fragment or variant of Pfu polymerasehaving polymerase activity.
 23. The method of claim 21, wherein thereaction mixture further comprises a polypeptide having 5′ to 3′exonuclease activity.
 24. The method of claim 1, wherein thethermostable DNA polymerase comprises a bacterial family A polymerase ora fragment or variant of a bacterial family A polymerase havingpolymerase activity.
 25. The method of claim 24, wherein thethermostable DNA polymerase comprises Taq DNA polymerase or a fragmentor variant of Taq DNA polymerase having polymerase activity. 26.(canceled)
 27. (canceled)
 28. The method of claim 25, wherein thethermostable DNA polymerase comprises a variant of Taq DNA polymerasehaving increased processivity relative to naturally occurring Taq DNApolymerase. 29-41. (canceled)
 42. A method of stabilizing an DNA:RNAduplex comprising combining the DNA:RNA duplex with a polypeptidecomprising an amino acid sequence of a nucleic acid binding polypeptideor a fragment thereof having nucleic acid binding activity. 43-90.(canceled)